Cancer Vaccines Targeting PRAME and Uses Thereof

ABSTRACT

Disclosed herein are nucleic acid molecules comprising one or more nucleic acid sequences that encode a mutated consensus PRAME antigen. Vectors, compositions, and vaccines comprising one or more nucleic acid sequences that encode a mutated consensus PRAME antigen are disclosed. Methods of treating a subject with a PRAME-expressing tumor and methods of preventing a PRAME-expressing tumor are disclosed. Mutated consensus PRAME antigen is disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. patentapplication Ser. No. 16/219,356, filed Dec. 13, 2018, and U.S.Provisional Patent Application No. 62/598,290, filed Dec. 13, 2017, thedisclosures of which are incorporated herein by reference in theirentirety.

SEQUENCE LISTING

This application contains a Sequence Listing which has been submittedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created Apr. 20, 2022, is named104409_000685_sequence_listing.txt and is 42,858 bytes in size.

TECHNICAL FIELD

The present invention relates to Preferentially Expressed Antigen inMelanoma (PRAME) antigens and nucleic acid molecules which encode thesame. The present invention also relates to vaccines including suchPRAME immunogens and/or nucleic acid molecules. The present inventionfurther relates to methods of using the vaccines for inducing immuneresponses and preventing and/or treating subjects having cancer cellsand tumors that express PRAME.

BACKGROUND

Cancer is among the leading causes of death worldwide and, in the UnitedStates, is the second most common cause of death, accounting for nearlyone of every four deaths. The cancer vaccine market is growing rapidly.Effective tumor vaccines may be useful to prevent tumor growth and/ormay be useful as being a more effective, less toxic alternative tostandard treatments for patients with advanced cancers. An antigenassociated with cancer and, therefore, a target for anti-tumor vaccinesis PRAME.

PRAME, originally identified as a gene encoding a HLA-A24 restrictedantigenic peptide in human melanoma, triggers autologous cytotoxic Tcell-medicated immune responses. The human PRAME gene, located onchromosome 22 (HSA22), encodes a protein with seven leucine-rich (LXXLL(SEQ ID NO: 3)) motifs through which PRAME interferes with the retinoicacid receptor (RAR) pathway, and leads to the inhibition of RA-induceddifferentiation, growth arrest, and apoptosis (Epping, M. T. et al. Thehuman tumor antigen PRAME is a dominant repressor of retinoic acidreceptor signaling. Cell 122, 835-847, doi:10.1016/j.cell.2005.07.003(2005)). In this way, PRAME functions as a transcriptional repressor ofsignaling pathways, and the over-expression of PRAME results intumorigenesis.

Because its expression is low or absent in almost all normal adulttissues except for testis, PRAME is considered a cancer testis antigen(CTA). In addition to melanoma, PRAME is overexpressed in a variety ofother human malignancies, including acute and chronic leukemia, multiplemyeloma, medulloblastoma, sarcomas, head and neck cancer, breast cancer,non-small cell lung cancer, renal and ovarian cancer. In a study ofovarian carcinoma, PRAME expression was identified in 100% of surgicalsamples (n=27) (Brenne, K., Nymoen, D. A., Reich, R. & Davidson, B.PRAME (preferentially expressed antigen of melanoma) is a novel markerfor differentiating serous carcinoma from malignant mesothelioma.American journal of clinical pathology 137, 240-247,doi:10.1309/AJCPGA95KVSAUDMF (2012)).

Prevention, diagnosis, and treatment of cancer may take many differentforms. Prevention may include screening for pre-disposing factors (e.g.,specific genetic variants), altering behavior (e.g., smoking, diet, andamount of physical activity), and vaccination against viruses (e.g.,human papilloma virus hepatitis B virus). Treatment may includechemotherapy, radiation therapy, and surgical removal of a tumor orcancerous tissue. Despite the availability of numerous prevention andtreatment methods, such methods often meet with limited success ineffectively preventing and/or treating the cancer.

Vaccines for the treatment and prevention of cancer are of interest.However, existing vaccines targeting tumor cell antigens are limited bypoor antigen expression in vivo. Accordingly, a need remains in the artfor safe and effective vaccines and methods of their use for preventingand/or treating cancer and reducing mortality in subjects suffering fromcancer.

SUMMARY OF THE INVENTION

The present disclosure is directed to nucleic acid molecules comprisingSEQ ID NO:1 and nucleic acid molecules encoding the amino acid sequenceset forth in SEQ ID NO:2. In some embodiments, the nucleic acid moleculecomprises the nucleic acid sequence set forth in SEQ ID NO: 1. Infurther embodiments, the nucleic acid molecules comprise the nucleicacid sequence set forth in nucleotides 55 to 1584 of SEQ ID NO: 1. Instill further embodiments, the nucleic acid molecule comprises a nucleicacid sequence that encodes the amino acid sequence set forth in SEQ IDNO: 2. In further embodiments, the nucleic acid molecule comprises anucleic acid sequence that encodes the amino acid sequence as set forthin amino acid residues 19 to 526 of SEQ ID NO: 2. In still furtherembodiments, the vector comprises the nucleic acid molecule of claim 1.

In still further embodiments, the nucleic acid molecules encode a PRAMEantigen. In some embodiments, the encoded PRAME antigen comprises theamino acid sequence set forth in amino acid residues 19 to 526 of SEQ IDNO: 2. In some embodiments, the encoded PRAME antigen comprises SEQ IDNO: 2.

The nucleic acid molecules described herein may be incorporated into avector, such as a plasmid or viral vector. In some embodiments, thevector comprises the nucleic acid sequence set forth in SEQ ID NO: 1. Incertain embodiments, the vector comprises the nucleic acid sequence setforth in nucleotides 55 to 1584 of SEQ ID NO: 1. In further embodiments,the vector comprises a nucleic acid sequence that encodes the amino acidsequence set forth in SEQ ID NO: 2. In still further embodiments, thevector comprises a nucleic acid sequence that encodes the amino acidsequence as set forth in amino acid residues 19 to 526 of SEQ ID NO: 2.In certain embodiments, the vector comprises the nucleic acid moleculeof claim 1.

In some embodiments, the nucleic acids described herein are operablylinked to a regulatory element. In some embodiments the regulatoryelement is a promoter and/or a poly-adenylation signal. In furtherembodiments, the promoter is a human cytomegalovirus immediate-earlypromoter (hCMV promoter). In still further embodiments, thepoly-adenylation signal is a bovine growth hormone poly-adenylationsignal (bGH polyA).

Further provided herein is a PRAME antigenic protein comprising theamino acid sequence set forth in amino acid residues 19 to 526 of SEQ IDNO: 2. In some embodiments, the PRAME antigen comprises SEQ ID NO: 2.

Vaccines comprising a PRAME antigen, wherein the antigen comprises theamino acid sequence set forth in amino acid residues 19 to 526 of SEQ IDNO: 2 are also provided. In some embodiments, the PRAME antigencomprises SEQ ID NO: 2. In some embodiments, the PRAME antigen isencoded by nucleotides 55 to 1584 of SEQ ID NO: 1. In some embodiments,the PRAME antigen is encoded by a nucleic acid molecule comprising SEQID NO: 1.

Also provided herein are vaccines comprising a nucleic acid moleculeencoding a disclosed PRAME antigen. In some embodiments, the encodedPRAME antigen comprises the amino acid sequence set forth in amino acidresidues 19 to 526 of SEQ ID NO: 2. In some embodiments, the encodedPRAME antigen comprises SEQ ID NO: 2. In some embodiments, the PRAMEantigen is encoded by nucleotides 55 to 1584 of SEQ ID NO: 1. In someembodiments, the PRAME antigen is encoded by a nucleic acid moleculecomprising SEQ ID NO: 1. In some embodiments, the nucleic acid moleculeis incorporated into a vector, including but not limited to a plasmid orviral vector.

The disclosed vaccines may further comprise a pharmaceuticallyacceptable excipient. In some embodiments, the vaccines may furthercomprise an adjuvant. In certain embodiments, the adjuvant is IL-12,IL-15, IL-28, or RANTES.

Further provided herein are methods for treating a subject having a cellcharacterized by aberrant PRAME expression comprising administering atherapeutically effective amount of the vaccine. In some embodiments,the administration includes an electroporation step. In otherembodiments, the administration occurs at one or more sites on thesubject.

Also described herein are methods of treating cancer in a subject, themethod comprising administering a therapeutically effective amount of avaccine to the subject. Methods are also provided for vaccinating asubject against cells characterized by aberrant PRAME expressioncomprising administering a vaccine in an amount effective to elicit animmune response. The vaccine administered in the methods taught in thisdisclosure comprise a nucleic acid as described above. In someembodiments, the administration includes an electroporation step. Inother embodiments, the administration occurs at one or more sites on thesubject.

In some embodiments, the nucleic acid molecules described herein are foruse as a medicament. In some embodiments, the nucleic acid moleculesdescribed herein are for use as a medicament in the treatment of cancer.In some embodiments, the nucleic acid molecules described herein are foruse in the preparation of a medicament. In some embodiments, the nucleicacid molecules described herein are for use in the preparation of amedicament for the treatment of cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and FIG. 1B depict schematic representation of PRAME. FIG. 1Aprovides a graphical representation of the location of amino acidresidue numbers of the seven putative nuclear receptor (NR) boxes (LXXLLmotifs (SEQ ID NO:3)) in PRAME protein. FIG. 1A discloses SEQ ID NOS58-63, and 4, respectively, in order of appearance.

FIG. 1B provides the amino acid sequence data, including adjacent aminoacid residues, for each NR box. FIG. 1B discloses SEQ ID NOS 3 and64-70, respectively, in order of appearance.

FIG. 2 provides a sequence alignment of a modified synthetic consensusPRAME with human PRAME sequences. FIG. 2 discloses SEQ ID NOS 71-74, and74, respectively, in order of appearance.

FIG. 3 generally depicts the cloning reaction that yields pGX1421.

FIG. 4 illustrates confirmation by Western blot analysis of expressionof PRAME antigen in cell lines transfected with the synthetic consensusPRAME.

FIG. 5 depicts results of an immunoassay comparing expression ofsynthetic consensus PRAME to a control (pVAX).

FIG. 6 depicts the immunization and bleed schedules for animmunogenicity study performed in C57Bl/6 mice.

FIG. 7 depicts the dose response of pGX1411 in mice.

FIG. 8A illustrates confirmation that pGX1411 is immunogenic in mice.

FIG. 8B graphically depicts the immune response in mice afteradministration of pGX1411 or untreated mice.

FIGS. 9A-9F graphically depict the results of flow cytometry analysis todetermine the CD4+ and CD8+ T-cell responses in mice. Specifically,FIGS. 9A and 9B show the CD4+ and CD8+ response for producing IFNγ inmice receiving pGX1411 compared to untreated controls, respectively.FIGS. 9C and 9D show the CD4+ and CD8+ response for producing CD107a+ inmice receiving pGX1411 compared to untreated controls, respectively.FIGS. 9E and 9F show the CD4+ and CD8+ response for producing TNFα inmice receiving pGX1411 compared to untreated controls, respectively.

FIG. 10 graphically depicts endpoint titers after treatment with 5, 25,and 50 μg of pGX1411 compared to untreated controls.

FIG. 11 presents immunohistochemistry staining of cancerous tissue.

FIG. 12 graphically illustrates the comparison of immune response inmice administered synthetic consensus PRAME and modified syntheticconsensus PRAME.

FIG. 13 illustrates the immunization and bleeding schedule for non-humanprimate (NHP) studies.

FIGS. 14A-14C depict cellular immunogenicity as determined by IFNγELISpot. FIG. 14A illustrates the mean IFNγ response with the immunizedgroup over time.

FIG. 14B illustrates the IFNγ responses for individual NHPs. FIG. 14Cillustrates the IFNγ responses in the groups along with variation withinthe group. Timing of administration of pGX1421 and pGX6006 is indicatedby arrows 1-4.

FIGS. 15A-15D illustrate the CD4+ and CD8+ T-cells responses in NHPsadministered pGX1421 and PGX6006. FIG. 15A graphically displays CD4+T-cell response. FIG. 15B graphically depicts CD8+ response. FIG. 15Cgraphically depicts CD8+GrB+ T-cell response. FIG. 15D illustrates theshift in CD8+ T-cell phenotype after immunization.

FIGS. 16A-16C illustrate the cellular immune responses induced bypGX1421 and pGX1421 in combination with pGX6006 (IL-12). FIG. 16Adepicts IFNγ response in individual NHPs administered pGX1421. FIG. 16Billustrates IFNγ response in individual NHPs administered both pGX1421and pGX6006. FIG. 16C provides a comparison of the responses depicted inFIGS. 16A and 16B. Timing of administration of pGX1421 and pGX1421 incombination with pGX6006 is indicated by arrows 1-4.

FIGS. 17A-17F illustrate the cellular immune responses induced bypGX1421 and pGX1421 in combination with pGX6006 as characterized by flowcytometry. FIG. 17A depicts minimal CD4+ response in any individualrecipient, and FIG. 17B illustrates the difference in INFγ and TNFαbetween the groups. FIG. 17C depicts a greater CD8+ T-cell response forpGX1421/pGX6006 administration rather than pGX1421 administration alone,and FIG. 17D illustrates the difference in INFγ and TNFα between thegroups. FIG. 17E depicts the CD8+GrB+ T-cell response in NHPsadministered pGX1421 alone or in combination with pGX6006, and FIG. 17Fillustrates the difference in INFγ and TNFα between the groups.

FIGS. 18A-18C compare IFNγ responses induced by administration ofpGX1421 in combination with pGX6006 (FIG. 18A) to those responsesinduced by administration of pGX1411 in combination with pGX6006 (FIG.18B). FIG. 18C combines the data of FIGS. 18A and 18B for ease ofcomparison. Timing of administration of pGX1421 in combination withpGX6006 and pGX1411 in combination with pGX6006 is indicated by arrows1-4.

FIGS. 19A-19F illustrate the cellular immune responses induced byadministration of pGX1421 in combination with pGX6006 and those sameresponses induced by administration of pGX1411 in combination withpGX6006. Specifically, FIG. 19A illustrates the CD4+ T-cell responsesinduced by administration of pGX1421 in combination with pGX6006 and theCD4+ T-cell responses induced by administration of pGX1411 incombination with pGX6006, and FIG. 19D illustrates in INFγ and TNFαbetween these two groups. FIG. 19B illustrates the CD8+ T-cell responsesinduced by administration of pGX1421 in combination with pGX6006 and theCD8+ T-cell responses induced by administration of pGX1411 incombination with pGX6006, and FIG. 19E illustrates in INFγ and TNFαbetween these two groups. FIG. 19C illustrates the CD8+GrB+ T-cellresponses induced by administration of pGX1421 in combination withpGX6006 and the CD8+GrB+ T-cell responses induced by administration ofpGX1411 in combination with pGX6006, and FIG. 19F illustrates in INFγand TNFα between the groups.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to vaccines comprising a PRAME antigen.The vaccines provide treatment and/or prevention for a cancer expressingPRAME. The vaccine of the invention can provide any combination ofparticular cancer antigens for the particular prevention or treatment ofthe cancer of a subject that is in need of treatment.

One manner for designing the nucleic acid and its encoded amino acidsequence of the recombinant cancer antigen is by introducing mutationsthat change particular amino acids in the overall amino acid sequence ofthe native cancer antigen. The introduction of mutations does not alterthe cancer antigen so much that it cannot be universally applied acrossa mammalian subject, and preferably a human or dog subject, but changesit enough that the resulting amino acid sequence breaks tolerance or isconsidered a foreign antigen in order to generate an immune response.Another manner may be creating a consensus recombinant cancer antigenthat has at least 85% and up to 99% amino acid sequence identity to itscorresponding native cancer antigen; preferably at least 90% and up to98% sequence identity; more preferably at least 93% and up to 98%sequence identity; or even more preferably at least 95% and up to 98%sequence identity. In some instances the recombinant cancer antigen is95%, 96%, 97%, 98%, or 99% amino acid sequence identity to itscorresponding native cancer antigen. The native cancer antigen is theantigen normally associated with the particular cancer or cancer tumor.Depending upon the cancer antigen, the consensus sequence of the cancerantigen can be across mammalian species or within subtypes of a speciesor across viral strains or serotypes. Some cancer antigens do not varygreatly from the wild type amino acid sequence of the cancer antigen.Some cancer antigens have nucleic acid/amino acid sequences that are sodivergent across species, that a consensus sequence cannot be generated.In these instances, a recombinant cancer antigen that will breaktolerance and generate an immune response is generated that has at least85% and up to 99% amino acid sequence identity to its correspondingnative cancer antigen; preferably at least 90% and up to 98% sequenceidentity; more preferably at least 93% and up to 98% sequence identity;or even more preferably at least 95% and up to 98% sequence identity. Insome instances the recombinant cancer antigen is 95%, 96%, 97%, 98%, or99% amino acid sequence identity to its corresponding native cancerantigen. The aforementioned approaches can be combined so that the finalrecombinant cancer antigen has a percent similarity to native cancerantigen amino acid sequence as discussed above.

The PRAME antigen of the present disclosure can be a synthetic consensusPRAME antigen derived from the amino acid or nucleic acid sequences ofPRAME from different species or from different isoforms within aspecies, and thus, the synthetic consensus PRAME antigen is non-native.The synthetic consensus PRAME antigen also comprises amino acidsubstitutions in the protein domain that interacts with or mediatesinteraction with retinoic acid receptor (RAR). Specifically, leucineamino acid residues at amino acid residues 487 and 488 may besubstituted for by valine residues. Additionally, the PRAME antigen maycomprise a Kozak regulatory sequence and/or an IgE leader sequence toenhance the expression and immunogenicity, respectively.

The recombinant PRAME can induce antigen-specific T cell and/or hightiter antibody responses, thereby inducing or eliciting an immuneresponse that is directed to or reactive against the cancer or tumorexpressing the antigen. In some embodiments, the induced or elicitedimmune response can be a cellular, humoral, or both cellular and humoralimmune responses. In some embodiments, the induced or elicited cellularimmune response can include induction or secretion of interferon-gamma(IFN-γ) and/or tumor necrosis factor alpha (TNF-α). In otherembodiments, the induced or elicited immune response can reduce orinhibit one or more immune suppression factors that promote growth ofthe tumor or cancer expressing the antigen, for example, but not limitedto, factors that down regulate MHC presentation, factors that upregulate antigen-specific regulatory T cells (Tregs), PD-L1, FasL,cytokines such as IL-10 and TFG-β, tumor associated macrophages, tumorassociated fibroblasts, soluble factors produced by immune suppressorcells, CTLA-4, PD-1, MDSCs, MCP-1, and an immune checkpoint molecule.

The vaccine may be combined further with antibodies to checkpointinhibitors such as PD-1 and PDL-1 to increase the stimulation of boththe cellular and humoral immune responses. Using anti-PD-1 or anti-PDL-1antibodies prevents PD-1 or PDL-1 from suppressing T-cell and/or B-cellresponses. Overall, by designing the cancer antigens to be recognized bythe immune system helps to overcome other forms of immune suppression bytumor cells, and these vaccines can be used in combination withsuppression or inhibition therapies (such as anti-PD-1 and anti-PDL-1antibody therapies) to further increase T-cell and/or B-cell responses.

Definitions

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. In case of conflict, the present document, includingdefinitions, will control. Preferred methods and materials are describedbelow, although methods and materials similar or equivalent to thosedescribed herein can be used in practice or testing of the presentinvention. All publications, patent applications, patents and otherreferences mentioned herein are incorporated by reference in theirentirety. The materials, methods, and examples disclosed herein areillustrative only and not intended to be limiting. The terminology usedherein is for the purpose of describing particular embodiments only andis not intended to be limiting.

The terms “comprise(s),” “include(s),” “having,” “has,” “can,”“contain(s),” and variants thereof, as used herein, are intended to beopen-ended transitional phrases, terms, or words that do not precludethe possibility of additional acts or structures. The singular forms“a,” “and” and “the” include plural references unless the contextclearly dictates otherwise. The present disclosure also contemplatesother embodiments “comprising,” “consisting of” and “consistingessentially of” the embodiments or elements presented herein, whetherexplicitly set forth or not.

For recitation of numeric ranges herein, each intervening value havingthe same degree of precision as the recited range minimum and maximum isexplicitly contemplated. For example, for the range of 6-9, the numbers7 and 8 are contemplated in addition to 6 and 9, and for the range6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9,and 7.0 are explicitly contemplated.

“Adjuvant” as used herein means any molecule added to the vaccinesdescribed herein to enhance the immunogenicity of the PRAME antigensantigens and/or the nucleic acid molecules encoding the antigens asdescribed herein described herein.

“Antibody” as used herein means an antibody of classes IgG, IgM, IgA,IgD, or IgE, or fragments, or derivatives thereof, including Fab,F(ab′)2, Fd, and single chain antibodies, diabodies, bispecificantibodies, bifunctional antibodies, and derivatives thereof. Theantibody can be an antibody isolated from the serum sample of a mammal,a polyclonal antibody, an affinity purified antibody, or any mixturethereof which exhibits sufficient binding specificity to a desiredepitope or a sequence derived therefrom.

“PRAME Antigen” refers to: proteins having mutated PRAME amino acidsequences including amino acid residues 19 to 526 of SEQ ID NO:2. PRAMEantigens may optionally include signal peptides such as those from otherproteins. For example, a PRAME antigen comprising a signal peptide mayinclude the amino acid sequence set forth in SEQ ID NO: 2.

“Coding sequence” or “encoding nucleic acid” as used herein means thenucleic acids (RNA or DNA molecule) that comprise a nucleotide sequencewhich encodes a protein. The coding sequence can further includeinitiation and termination signals operably linked to regulatoryelements including a promoter and polyadenylation signal capable ofdirecting expression in the cells of a subject or mammal to which thenucleic acid is administered.

“Complement” or “complementary” as used herein means a nucleic acid canmean Watson-Crick (e.g., A-T/U and C-G) or Hoogsteen base pairingbetween nucleotides or nucleotide analogs of nucleic acid molecules.

“Consensus” or “consensus sequence” as used herein means a polypeptidesequence based on analysis of an alignment of multiple sequences for thesame gene from different organisms. Nucleic acid sequences that encode aconsensus polypeptide sequence can be prepared. Vaccines comprisingproteins that comprise consensus sequences and/or nucleic acid moleculesthat encode such proteins can be used to induce broad immunity againstan antigen.

“Constant current” as used herein describes a current that is receivedor experienced by a tissue, or cells defining said tissue, over theduration of an electrical pulse delivered to same tissue. The electricalpulse is delivered from the electroporation devices described herein.This current remains at a constant amperage in said tissue over the lifeof an electrical pulse because the electroporation device providedherein has a feedback element, preferably having instantaneous feedback.The feedback element can measure the resistance of the tissue (or cells)throughout the duration of the pulse and cause the electroporationdevice to alter its electrical energy output (e.g., increase voltage) socurrent in same tissue remains constant throughout the electrical pulse(on the order of microseconds), and from pulse to pulse. In someembodiments, the feedback element comprises a controller.

“Current feedback” or “feedback” as used herein may be usedinterchangeably and may mean the active response of the providedelectroporation devices, which comprises measuring the current in tissuebetween electrodes and altering the energy output delivered by the EPdevice accordingly in order to maintain the current at a constant level.This constant level is preset by a user prior to initiation of a pulsesequence or electrical treatment. The feedback may be accomplished bythe electroporation component, e.g., controller, of the electroporationdevice, as the electrical circuit therein is able to continuouslymonitor the current in tissue between electrodes and compare thatmonitored current (or current within tissue) to a preset current andcontinuously make energy-output adjustments to maintain the monitoredcurrent at preset levels. The feedback loop may be instantaneous as itis an analog closed-loop feedback.

“Decentralized current” as used herein may mean the pattern ofelectrical currents delivered from the various needle electrode arraysof the electroporation devices described herein, wherein the patternsminimize, or preferably eliminate, the occurrence of electroporationrelated heat stress on any area of tissue being electroporated.

“Electroporation,” “electro-permeabilization,” or “electro-kineticenhancement” (“EP”) as used interchangeably herein means the use of atransmembrane electric field pulse to induce microscopic pathways(pores) in a bio-membrane; their presence allows biomolecules such asplasmids, oligonucleotides, siRNA, drugs, ions, and water to pass fromone side of the cellular membrane to the other.

“Fragment” as used herein with respect to nucleic acid sequences means anucleic acid sequence or a portion thereof, that encodes a polypeptidecapable of eliciting an immune response in a mammal that cross reactswith an antigen disclosed herein. The fragments can be DNA fragmentsselected from at least one of the various nucleotide sequences thatencode protein fragments set forth below. Fragments can comprise atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, or at least 95% ofone or more of the nucleic acid sequences set forth below, excluding anheterologous signal peptide added. The fragment may comprise at least95%, at least 96%, at least 97%, at least 98%, or at least 99% of one ormore of the nucleic acid sequences set forth below and additionallyoptionally comprise sequence encoding a heterologous signal peptidewhich is not included when calculating percent identity. Fragments mayfurther comprise coding sequences for a signal peptide such as animmunoglobulin signal peptide, for example an IgE or IgG signal peptide.The coding sequence encoding an N terminal methionine and/or signalpeptide may be linked to a fragment of coding sequence.

In some embodiments, fragments can comprise at least 20 nucleotides ormore, at least 30 nucleotides or more, at least 40 nucleotides or more,at least 50 nucleotides or more, at least 60 nucleotides or more, atleast 70 nucleotides or more, at least 80 nucleotides or more, at least90 nucleotides or more, at least 100 nucleotides or more, at least 150nucleotides or more, at least 200 nucleotides or more, at least 250nucleotides or more, at least 300 nucleotides or more, at least 350nucleotides or more, at least 400 nucleotides or more, at least 450nucleotides or more, at least 500 nucleotides or more, at least 550nucleotides or more, at least 600 nucleotides or more, at least 650nucleotides or more, at least 700 nucleotides or more, at least 750nucleotides or more, at least 800 nucleotides or more, at least 850nucleotides or more, at least 900 nucleotides or more, at least 950nucleotides or more, or at least 1000 nucleotides or more of at leastone of the nucleic acid sequences set forth below.

“Fragment” or “immunogenic fragment” with respect to polypeptidesequences means a polypeptide capable of eliciting an immune response ina mammal that cross reacts with an antigen disclosed herein. Thefragments can be polypeptide fragments selected from at least one of thevarious amino acids sequences below. Fragments of consensus proteins cancomprise at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90% or atleast 95% of a consensus protein, excluding any heterologous signalpeptide added. The fragment may comprise at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% of one or more of the aminosequences set forth below and additionally optionally comprise aheterologous signal peptide which is not included when calculatingpercent identity. Fragments may further comprise a signal peptide suchas an immunoglobulin signal peptide, for example an IgE or IgG signalpeptide.

In some embodiments, fragments of consensus proteins can comprise atleast 20 amino acids or more, at least 30 amino acids or more, at least40 amino acids or more, at least 50 amino acids or more, at least 60amino acids or more, at least 70 amino acids or more, at least 80 aminoacids or more, at least 90 amino acids or more, at least 100 amino acidsor more, at least 110 amino acids or more, at least 120 amino acids ormore, at least 130 amino acids or more, at least 140 amino acids ormore, at least 150 amino acids or more, at least 160 amino acids ormore, at least 170 amino acids or more, at least 180 amino acids or moreof a protein sequence disclosed herein.

As used herein, the term “genetic construct” refers to the DNA or RNAmolecules that comprise a nucleotide sequence that encodes a protein.The coding sequence includes initiation and termination signals operablylinked to regulatory elements including a promoter and polyadenylationsignal capable of directing expression in the cells of the subject towhom the nucleic acid molecule is administered. As used herein, the term“expressible form” refers to a gene construct that contain the necessaryregulatory elements operably linked to a coding sequence that encodes aprotein such that, when present in cell of a subject, the codingsequence will be expressed.

The term “homology,” as used herein, refers to a degree ofcomplementarity. There can be partial homology or complete homology(i.e., identity). A partially complementary sequence that at leastpartially inhibits a completely complementary sequence from hybridizingto a target nucleic acid is referred to using the functional term“substantially homologous.” When used in reference to a double-strandednucleic acid sequence such as a cDNA or genomic clone, the term“substantially homologous,” as used herein, refers to a probe that canhybridize to a strand of the double-stranded nucleic acid sequence underconditions of low stringency. When used in reference to asingle-stranded nucleic acid sequence, the term “substantiallyhomologous,” as used herein, refers to a probe that can hybridize to(i.e., is the complement of) the single-stranded nucleic acid templatesequence under conditions of low stringency.

“Identical” or “identity” as used herein in the context of two or morenucleic acids or polypeptide sequences means that the sequences have aspecified percentage of residues that are the same over a specifiedregion. The percentage can be calculated by optimally aligning the twosequences, comparing the two sequences over the specified region,determining the number of positions at which the identical residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the specified region, and multiplying the result by 100 toyield the percentage of sequence identity. In cases where the twosequences are of different lengths or the alignment produces one or morestaggered ends and the specified region of comparison includes only asingle sequence, the residues of single sequence are included in thedenominator but not the numerator of the calculation. When comparing DNAand RNA, thymine (T) and uracil (U) can be considered equivalent.Identity can be performed manually or by using a computer sequencealgorithm such as BLAST or BLAST 2.0.

“Impedance” as used herein may be used when discussing the feedbackmechanism and can be converted to a current value according to Ohm'slaw, thus enabling comparisons with the preset current.

“Immune response” as used herein means the activation of a host's immunesystem, e.g., that of a mammal, in response to the introduction ofantigen. The immune response can be in the form of a cellular or humoralresponse, or both.

“Nucleic acid” or “oligonucleotide” or “polynucleotide” as used hereinmeans at least two nucleotides covalently linked together. The depictionof a single strand also defines the sequence of the complementarystrand. Thus, a nucleic acid also encompasses the complementary strandof a depicted single strand. Many variants of a nucleic acid can be usedfor the same purpose as a given nucleic acid. Thus, a nucleic acid alsoencompasses substantially identical nucleic acids and complementsthereof. A single strand provides a probe that can hybridize to a targetsequence under stringent hybridization conditions. Thus, a nucleic acidalso encompasses a probe that hybridizes under stringent hybridizationconditions.

Nucleic acids can be single stranded or double stranded, or can containportions of both double stranded and single stranded sequence. Thenucleic acid can be DNA, both genomic and cDNA, RNA, or a hybrid, wherethe nucleic acid can contain combinations of deoxyribo- andribo-nucleotides, and combinations of bases including uracil, adenine,thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosineand isoguanine. Nucleic acids can be obtained by chemical synthesismethods or by recombinant methods.

“Operably linked” as used herein means that expression of a gene isunder the control of a promoter with which it is spatially connected. Apromoter can be positioned 5′ (upstream) or 3′ (downstream) of a geneunder its control. The distance between the promoter and a gene can beapproximately the same as the distance between that promoter and thegene it controls in the gene from which the promoter is derived. As isknown in the art, variation in this distance can be accommodated withoutloss of promoter function.

A “peptide,” “protein,” or “polypeptide” as used herein can mean alinked sequence of amino acids and can be natural, synthetic, or amodification or combination of natural and synthetic.

“Promoter” as used herein means a synthetic or naturally derivedmolecule which is capable of conferring, activating, or enhancingexpression of a nucleic acid in a cell. A promoter can comprise one ormore specific transcriptional regulatory sequences to further enhanceexpression and/or to alter the spatial expression and/or temporalexpression of a nucleic acid in a cell. A promoter can also comprisedistal enhancer or repressor elements, which can be located as much asseveral thousand base pairs from the start site of transcription. Apromoter can be derived from sources including viral, bacterial, fungal,plant, insect, and animal. A promoter can regulate the expression of agene component constitutively or differentially with respect to cell,tissue, or organ in which expression occurs, or with respect to thedevelopmental stage at which expression occurs, or in response toexternal stimuli such as physiological stresses, pathogens, metal ions,or inducing agents. Representative examples of promoters include thebacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lacoperator-promoter, tac promoter, SV40 late promoter, SV40 earlypromoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40late promoter and the CMV IE promoter.

“Signal peptide” and “leader sequence” are used interchangeably hereinand refer to an amino acid sequence that can be linked at the aminoterminus of a protein set forth herein. Signal peptides/leader sequencestypically direct localization of a protein. Signal peptides/leadersequences used herein preferably facilitate secretion of the proteinfrom the cell in which it is produced. Signal peptides/leader sequencesare often cleaved from the remainder of the protein, often referred toas the mature protein, upon secretion from the cell. Signalpeptides/leader sequences are linked at the amino terminus (i.e., Nterminus) of the protein.

“Stringent hybridization conditions” as used herein means conditionsunder which a first nucleic acid sequence (e.g., probe) will hybridizeto a second nucleic acid sequence (e.g., target), such as in a complexmixture of nucleic acids. Stringent conditions are sequence-dependentand will be different in different circumstances. Stringent conditionscan be selected to be about 5-10° C. lower than the thermal meltingpoint (Tm) for the specific sequence at a defined ionic strength pH. TheTm can be the temperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at Tm, 50% of the probes are occupied atequilibrium). Stringent conditions can be those in which the saltconcentration is less than about 1.0 M sodium ion, such as about0.01-1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3and the temperature is at least about 30° C. for short probes (e.g.,about 10-50 nucleotides) and at least about 60° C. for long probes(e.g., greater than about 50 nucleotides). Stringent conditions can alsobe achieved with the addition of destabilizing agents such as formamide.For selective or specific hybridization, a positive signal can be atleast 2 to 10 times background hybridization. Exemplary stringenthybridization conditions include the following: 50% formamide, 5×SSC,and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS, incubating at 65°C., with wash in 0.2×SSC, and 0.1% SDS at 65° C.

“Subject” as used herein can mean a mammal that wants to or is in needof being immunized with the herein described vaccines. The mammal can bea human, chimpanzee, dog, cat, horse, cow, mouse, or rat.

“Substantially complementary” as used herein means that a first sequenceis at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identicalto the complement of a second sequence over a region of 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540, ormore nucleotides or amino acids, or that the two sequences hybridizeunder stringent hybridization conditions.

“Substantially identical” as used herein means that a first and secondsequence are at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 180, 270, 360, 450, 540 or more nucleotides or amino acids,or with respect to nucleic acids, if the first sequence is substantiallycomplementary to the complement of the second sequence.

“Treat,” “treatment,” or “treating” as used herein can mean protectingan animal from a disease through means of preventing, suppressing,repressing, or completely eliminating the disease. Preventing thedisease involves administering a vaccine of the present invention to ananimal prior to onset of the disease. Suppressing the disease involvesadministering a vaccine of the present invention to an animal afterinduction of the disease but before its clinical appearance. Repressingthe disease involves administering a vaccine of the present invention toan animal after clinical appearance of the disease.

“Variant” as used herein with respect to a nucleic acid means (i) aportion or fragment of a referenced nucleotide sequence; (ii) thecomplement of a referenced nucleotide sequence or portion thereof; (iii)a nucleic acid that is substantially identical to a referenced nucleicacid or the complement thereof; or (iv) a nucleic acid that hybridizesunder stringent conditions to the referenced nucleic acid, complementthereof, or a sequence substantially identical thereto.

“Variant” as used herein with respect to a peptide or polypeptide meansa peptide or polypeptide that differs in amino acid sequence by theinsertion, deletion, or conservative substitution of amino acids, butretains at least one biological activity. Variant can also mean aprotein with an amino acid sequence that is substantially identical to areferenced protein with an amino acid sequence that retains at least onebiological activity. A conservative substitution of an amino acid, i.e.,replacing an amino acid with a different amino acid of similarproperties (e.g., hydrophilicity, degree and distribution of chargedregions) is recognized in the art as typically involving a minor change.These minor changes can be identified, in part, by considering thehydropathic index of amino acids, as understood in the art. Kyte et al.,J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an amino acidis based on a consideration of its hydrophobicity and charge. It isknown in the art that amino acids of similar hydropathic indexes can besubstituted and still retain protein function. In one aspect, aminoacids having hydropathic indexes of ±2 are substituted. Thehydrophilicity of amino acids can also be used to reveal substitutionsthat would result in proteins retaining biological function. Aconsideration of the hydrophilicity of amino acids in the context of apeptide permits calculation of the greatest local average hydrophilicityof that peptide, a useful measure that has been reported to correlatewell with antigenicity and immunogenicity. U.S. Pat. No. 4,554,101,incorporated fully herein by reference. Substitution of amino acidshaving similar hydrophilicity values can result in peptides retainingbiological activity, for example immunogenicity, as is understood in theart. Substitutions can be performed with amino acids havinghydrophilicity values within ±2 of each other. Both the hydrophobicityindex and the hydrophilicity value of amino acids are influenced by theparticular side chain of that amino acid. Consistent with thatobservation, amino acid substitutions that are compatible withbiological function are understood to depend on the relative similarityof the amino acids, and particularly the side chains of those aminoacids, as revealed by the hydrophobicity, hydrophilicity, charge, size,and other properties.

A variant may be a nucleic acid sequence that is substantially identicalover the full length of the full gene sequence or a fragment thereof.The nucleic acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical over the full length of the gene sequence or a fragmentthereof. A variant may be an amino acid sequence that is substantiallyidentical over the full length of the amino acid sequence or fragmentthereof. The amino acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% identical over the full length of the amino acid sequence or afragment thereof.

“Vector” as used herein means a nucleic acid sequence containing anorigin of replication. A vector can be a viral vector, bacteriophage,bacterial artificial chromosome, or yeast artificial chromosome. Avector can be a DNA or RNA vector. A vector can be a self-replicatingextrachromosomal vector, and preferably, is a DNA plasmid. The vectorcan contain or include one or more heterologous nucleic acid sequences.

Vaccine

Provided herein are vaccines comprising a herein-described PRAME antigenor a nucleic acid molecule encoding such an antigen. In someembodiments, the PRAME antigen comprises the amino acid sequence setforth in amino acid residues 19 to 526 of SEQ ID NO: 2. In someembodiments, the nucleic acid molecule encodes a PRAME antigen havingthe amino acid sequence set forth in amino acid residues 19 to 526 SEQID NO:2. In some embodiments, the PRAME antigen comprises the amino acidsequence set forth in SEQ ID NO: 2. In some embodiments, the nucleicacid molecule encodes a PRAME antigen having the amino acid sequence setforth SEQ ID NO:2. In some embodiments, the nucleic acid moleculeencoding the PRAME antigen comprises the nucleic acid sequence set forthin nucleotides 55 to 1584 of SEQ ID NO: 1. In some embodiments, thenucleic acid molecule encoding the PRAME antigen comprises the nucleicacid sequence set forth in SEQ ID NO: 1. The vaccines can be capable ofgenerating in a subject an immune response against the antigen. Theimmune response can be a therapeutic or prophylactic immune response.The vaccines may comprise a vector or a plurality of vectors asdescribed in more detail below.

The vaccines can be used to protect against cancer, for example, acancer or tumor expressing PRAME. The vaccines can be used to preventand/or treat an ovarian cancer expressing PRAME in a subject in needthereof. The vaccines can induce cellular and/or antibody responsesagainst PRAME and against cancers expressing PRAME in a subject in needthereof. In some embodiments of the present disclosure, the vaccine canbe used to protect against, prevent, treat, and/or induce cellularand/or antibody responses against cells characterized by aberrantexpression of PRAME. In some embodiments of the present disclosure, thevaccine can be used to protect against, prevent, treat, and/or inducecellular and/or antibody responses against ovarian cancer cellscharacterized by aberrant expression of PRAME, specifically epithelialovarian cancer cells, and more specifically, serous ovarian cancercells.

The development of a cancer vaccine as described herein comprisesidentifying a cancer antigen, e.g., PRAME, that is not recognized by theimmune system and is a self-antigen. The cancer antigen identified ischanged from a self-antigen to a foreign antigen in order to berecognized by the immune system. The redesign of the nucleic acid andamino acid sequence of the recombinant cancer antigen from a self to aforeign antigen breaks tolerance of the antigen by the immune system. Inorder to break tolerance, several redesign measures can be employed toproduce the cancer antigen as described below.

The breaking of tolerance can induce antigen-specific T cell and/or hightiter antibody responses, thereby inducing or eliciting an immuneresponse that is directed to or reactive against the cancer or tumorexpressing the antigen. In some embodiments, the induced or elicitedimmune response can be a cellular, humoral, or both cellular and humoralimmune responses. In some embodiments, the induced or elicited cellularimmune response can include induction or secretion of interferon-gamma(IFN-γ) and/or tumor necrosis factor alpha (TNF-α). In otherembodiments, the induced or elicited immune response can reduce orinhibit one or more immune suppression factors that promote growth ofthe tumor or cancer expressing the antigen, for example, but not limitedto, factors that downregulate MHC presentation, factors that upregulateantigen-specific regulatory T cells (Tregs), PD-L1, FasL, cytokines suchas IL-10 and TFG-β, tumor associated macrophages, tumor associatedfibroblasts, soluble factors produced by immune suppressor cells,CTLA-4, PD-1, MDSCs, MCP-1, and an immune checkpoint molecule.

In a particular embodiment, the vaccine can mediate clearance or preventgrowth of tumor cells by (1) increasing cytotoxic T lymphocyte such asCD8⁺ (CTL) to attack and kill tumor cells; (2) increasing T helper cellresponses; and/or (3) increasing inflammatory responses via IFN-γ andTFN-α, or preferably all of the aforementioned. The vaccine can increasetumor-free survival by 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%,40%, 41%, 42%, 43%, 44%, and 45%. The vaccine can reduce tumor mass by30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%,44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, and 60% after immunization. The vaccine can prevent and blockincreases in monocyte chemoattractant protein 1 (MCP-1), a cytokinesecreted by myeloid derived suppressor cells. The vaccine can increasetumor survival by 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%,41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, and 60%.

The vaccine can increase a cellular immune response in a subjectadministered the vaccine by about 50-fold to about 6000-fold, about50-fold to about 5500-fold, about 50-fold to about 5000-fold, about50-fold to about 4500-fold, about 100-fold to about 6000-fold, about150-fold to about 6000-fold, about 200-fold to about 6000-fold, about250-fold to about 6000-fold, or about 300-fold to about 6000-fold ascompared to a cellular immune response in a subject not administered thevaccine. In some embodiments the vaccine can increase the cellularimmune response in the subject administered the vaccine by about50-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold,400-fold, 450-fold, 500-fold, 550-fold, 600-fold, 650-fold, 700-fold,750-fold, 800-fold, 850-fold, 900-fold, 950-fold, 1000-fold, 1100-fold,1200-fold, 1300-fold, 1400-fold, 1500-fold, 1600-fold, 1700-fold,1800-fold, 1900-fold, 2000-fold, 2100-fold, 2200-fold, 2300-fold,2400-fold, 2500-fold, 2600-fold, 2700-fold, 2800-fold, 2900-fold,3000-fold, 3100-fold, 3200-fold, 3300-fold, 3400-fold, 3500-fold,3600-fold, 3700-fold, 3800-fold, 3900-fold, 4000-fold, 4100-fold,4200-fold, 4300-fold, 4400-fold, 4500-fold, 4600-fold, 4700-fold,4800-fold, 4900-fold, 5000-fold, 5100-fold, 5200-fold, 5300-fold,5400-fold, 5500-fold, 5600-fold, 5700-fold, 5800-fold, 5900-fold, or6000-fold as compared to the cellular immune response in the subject notadministered the vaccine.

The vaccine can increase interferon gamma (IFN-γ) levels in a subjectadministered the vaccine by about 50-fold to about 6000-fold, about50-fold to about 5500-fold, about 50-fold to about 5000-fold, about50-fold to about 4500-fold, about 100-fold to about 6000-fold, about150-fold to about 6000-fold, about 200-fold to about 6000-fold, about250-fold to about 6000-fold, or about 300-fold to about 6000-fold ascompared to IFN-γ levels in a subject not administered the vaccine. Insome embodiments the vaccine can increase IFN-γ levels in the subjectadministered the vaccine by about 50-fold, 100-fold, 150-fold, 200-fold,250-fold, 300-fold, 350-fold, 400-fold, 450-fold, 500-fold, 550-fold,600-fold, 650-fold, 700-fold, 750-fold, 800-fold, 850-fold, 900-fold,950-fold, 1000-fold, 1100-fold, 1200-fold, 1300-fold, 1400-fold,1500-fold, 1600-fold, 1700-fold, 1800-fold, 1900-fold, 2000-fold,2100-fold, 2200-fold, 2300-fold, 2400-fold, 2500-fold, 2600-fold,2700-fold, 2800-fold, 2900-fold, 3000-fold, 3100-fold, 3200-fold,3300-fold, 3400-fold, 3500-fold, 3600-fold, 3700-fold, 3800-fold,3900-fold, 4000-fold, 4100-fold, 4200-fold, 4300-fold, 4400-fold,4500-fold, 4600-fold, 4700-fold, 4800-fold, 4900-fold, 5000-fold,5100-fold, 5200-fold, 5300-fold, 5400-fold, 5500-fold, 5600-fold,5700-fold, 5800-fold, 5900-fold, or 6000-fold as compared to IFN-γlevels in the subject not administered the vaccine.

The vaccine can be a DNA vaccine. DNA vaccines are disclosed in U.S.Pat. Nos. 5,593,972; 5,739,118; 5,817,637; 5,830,876; 5,962,428;5,981,505; 5,580,859; 5,703,055; and 5,676,594, which are incorporatedherein fully by reference. The DNA vaccine can further comprise elementsor reagents that inhibit it from integrating into the chromosome.

The vaccine can include an RNA encoding the cancer antigen. The RNAvaccine can be introduced into the cell.

The vaccine can be an attenuated live vaccine, a vaccine usingrecombinant vectors to deliver antigen, subunit vaccines, andglycoprotein vaccines, for example, but not limited to, the vaccinesdescribed in U.S. Pat. Nos. 4,510,245; 4,797,368; 4,722,848; 4,790,987;4,920,209; 5,017,487; 5,077,044; 5,110,587; 5,112,749; 5,174,993;5,223,424; 5,225,336; 5,240,703; 5,242,829; 5,294,441; 5,294,548;5,310,668; 5,387,744; 5,389,368; 5,424,065; 5,451,499; 5,453,364;5,462,734; 5,470,734; 5,474,935; 5,482,713; 5,591,439; 5,643,579;5,650,309; 5,698,202; 5,955,088; 6,034,298; 6,042,836; 6,156,319 and6,589,529, which are each incorporated herein by reference.

In some embodiments, the nucleic acid vaccine may further comprisecoding sequence for a molecular adjuvant, in some cases the molecularadjuvant can be IL-12, IL-15, IL-28, IL-31, IL-33, and/or RANTES, and insome cases the molecular adjuvant is a checkpoint inhibitor, includinganti-cytotoxic T-lymphocyte antigen 4 (CTLA-4), anti-programmed deathreceptor-1 (PD-1) and anti-lymphocyte-activation gene (LAG-3). Codingsequence for IL-12, IL-15, IL-28, IL-31, IL-33 and/or RANTES may beincluded on one or more nucleic acid molecules that comprise codingsequence for one or more antigens. Coding sequence for IL-12, IL-15,IL-28, IL-31, IL-33, and/or RANTES may be included on a separate nucleicacid molecules such as a separate plasmid.

The vaccine of the present invention can have features required ofeffective vaccines such as being safe so that the vaccine itself doesnot cause illness or death; being protective against illness; inducingneutralizing antibody; inducing protective T cell responses; andproviding ease of administration, few side effects, biologicalstability, and low cost per dose. The vaccine can accomplish some or allof these features by containing the cancer antigen as discussed below.

The vaccine can further comprise one or more inhibitors of one or moreimmune checkpoint molecules (i.e., an immune checkpoint inhibitor).Immune checkpoint molecules are described below in more detail. Theimmune checkpoint inhibitor is any nucleic acid or protein that preventsthe suppression of any component in the immune system such as MHC classpresentation, T cell presentation and/or differentiation, B cellpresentation and/or differentiation, any cytokine, chemokine orsignaling for immune cell proliferation and/or differentiation. As alsodescribed below in more detail, the vaccine may be combined further withantibodies to checkpoint inhibitors such as PD-1 and PDL-1 to increasethe stimulation of both the cellular and humoral immune responses. Usinganti-PD-1 or anti-PDL-1 antibodies prevents PD-1 or PDL-1 fromsuppressing T-cell and/or B-cell responses.

Antigen

As described above, the vaccine can comprise an antigen or a nucleicacid molecule encoding an antigen. The antigen can be PRAME, a fragmentthereof, a variant thereof, or a combination thereof. PRAME is expressedin testis but not typically, or in relatively small amounts, in normal,noncancerous tissues. The PRAME protein is a repressor of retinoic acidreceptor, and without being bound to theory, this repression is thoughtto confer a growth advantage to cancer cells by repressing retinoic acidinduced arrest of cell proliferation and apoptosis. For example, PRAMEhas been associated with several forms of cancer and is a known cancerantigen. PRAME expression is increased in endometrial cancer, testiscancer, melanoma, and ovarian cancer.

Accordingly, the vaccine can be used for treating subjects sufferingfrom PRAME-expressing cancer. The vaccine can also be used for treatingsubjects with cancers or tumors that express PRAME or preventingdevelopment of such tumors in subjects. The PRAME antigen of the presentdisclosure differs from the native, “normal” PRAME antigen, and thusprovides therapy or prophylaxis against a PRAME antigen-expressingtumor. Accordingly, PRAME antigen sequences that differ from the nativePRAME gene (i.e., variant PRAME genes or sequences) are provided herein.Some aspects of the present invention provide for vaccine comprising anucleic acid molecule comprising the nucleic acid sequence set forth inSEQ ID NO: 1, and some aspects provide for a vaccine comprising anucleic acid molecule comprising a nucleic acid sequence that encodesthe amino acid sequence set forth in SEQ ID NO: 2.

Isolated nucleic acid molecules comprising the above-describedheterologous sequences are provided. Isolated nucleic acid moleculesconsisting of the above-described heterologous sequences are provided.Isolated nucleic acid molecules comprising the above-describedheterologous sequences may be incorporated into vectors such asplasmids, viral vectors and other forms of nucleic acid molecules asdescribed below. Thus, in some embodiments of the present disclosure,nucleic acid molecule is incorporated into a plasmid. In otherembodiments the nucleic acid molecule is incorporated into a vector.Some aspects of the present disclosure provide compositions comprisingthe nucleic acid having the nucleotide acid sequence SEQ ID NO:1 orhaving a nucleotide sequence encoding the amino acid sequence of SEQ IDNO:2.

Provided herein are nucleic acid molecules having sequences that encodePRAME antigens. In some embodiments, the nucleic acid molecule isincorporated into a vector, including but not limited to a plasmid or aviral vector. Coding sequences encoding PRAME antigens have thesequences as described above.

Protein molecules comprising the above described heterologous amino acidsequences are provided. Protein molecules consisting of the abovedescribed heterologous amino acid sequences are provided. Providedherein are proteins and polypeptides having the above-describedsequences. The proteins and polypeptide of the present disclosure may bereferred to as PRAME antigens and PRAME immunogens. PRAME antigens arecapable of eliciting an immune response against cancers and/or tumorsexpressing a PRAME antigen.

In one aspect, it is desired that the consensus antigen provides forimproved transcription and translation, including having one or more ofthe following: low GC content leader sequence to increase transcription;mRNA stability and codon optimization; and, to the extent possible,elimination of cis-acting sequence motifs (i.e., internal TATA-boxes).

In some aspects, it is desired to generate a consensus antigen thatgenerates a broad immune response across multiple strains, the consensusantigen having one or more of the following: incorporate all availablefull-length sequences; computer generated sequences that utilize themost commonly occurring amino acid at each position; and increasecross-reactivity between strains.

The PRAME antigen can be a consensus antigen (or immunogen) sequencederived from two or more species. The PRAME antigen can comprise aconsensus sequence and/or modification(s) for improved expression.Modification can include codon optimization, RNA optimization, additionof a Kozak sequence (e.g., GCC ACC) for increased translation initiationand/or the addition of an immunoglobulin leader sequence to increase theimmunogenicity of the PRAME antigen. The PRAME antigen can comprise asignal peptide such as an immunoglobulin signal peptide, for example,but not limited to, an immunoglobulin E (IgE) or immunoglobulin G (IgG)signal peptide. In some embodiments, the PRAME consensus antigen cancomprise a hemagglutinin (HA) tag. The PRAME consensus antigen can bedesigned to elicit stronger and broader cellular and/or humoral immuneresponses than a corresponding codon optimized PRAME antigen.

The PRAME consensus antigen can comprise one or more variants in one ormore functional domains of the protein, thereby eliciting stronger andbroader cellular and/or humoral immune responses than a correspondingcodon optimized PRAME antigen. The one or more mutations can be asubstitution of one or more of the amino acids in a domain of the PRAMEprotein that mediates interaction with RAR.

Vaccine in Combination with Immune Checkpoint Inhibitor

The vaccine can further comprise one or more inhibitors of one or moreimmune checkpoint molecules (i.e., an immune checkpoint inhibitor).Immune check point molecules are described below in more detail. Theimmune checkpoint inhibitor is any nucleic acid or protein that preventsthe suppression of any component in the immune system such as MHC classpresentation, T cell presentation and/or differentiation, B cellpresentation and/or differentiation, any cytokine, chemokine orsignaling for immune cell proliferation and/or differentiation.

Such an inhibitor can be a nucleic acid sequence, an amino acidsequence, a small molecule, or a combination thereof. The nucleic acidsequence can be DNA, RNA, cDNA, a variant thereof, a fragment thereof,or a combination thereof. The nucleic acid can also include additionalsequences that encode linker or tag sequences that are linked to theimmune checkpoint inhibitor by a peptide bond. The small molecule may bea low molecular weight, for example, less than 800 Daltons, organic orinorganic compound that can serve as an enzyme substrate, ligand (oranalog thereof) bound by a protein or nucleic acid, or regulator of abiological process. The amino acid sequence can be protein, a peptide, avariant thereof, a fragment thereof, or a combination thereof.

In some embodiments, the immune checkpoint inhibitor can be one or morenucleic acid sequences encoding an antibody, a variant thereof, afragment thereof, or a combination thereof. In other embodiments, theimmune check point inhibitor can be an antibody, a variant thereof, afragment thereof, or a combination thereof

1. Immune Checkpoint Molecule

The immune check point molecule can be a nucleic acid sequence, an aminoacid sequence, a small molecule, or a combination thereof. The nucleicacid sequence can be DNA, RNA, cDNA, a variant thereof, a fragmentthereof, or a combination thereof. The nucleic acid can also includeadditional sequences that encode linker or tag sequences that are linkedto the immune checkpoint inhibitor by a peptide bond. The small moleculemay be a low molecular weight, for example, less than 800 Daltons,organic or inorganic compound that can serve as an enzyme substrate,ligand (or analog thereof) bound by a protein or nucleic acid, orregulator of a biological process. The amino acid sequence can beprotein, a peptide, a variant thereof, a fragment thereof, or acombination thereof.

a. PD-1 and PD-L1

The immune checkpoint molecule may programmed cell death protein 1(PD-1), programmed cell death ligand 1 (PD-L1), a fragment thereof, avariant thereof, or a combination thereof. PD-1 is a cell surfaceprotein encoded by the PDCD1 gene. PD-1 is a member of theimmunoglobulin superfamily and is expressed on T cells and pro-B cells,and thus, contributes to the fate and/or differentiation of these cells.In particular, PD-1 is a type 1 membrane protein of the CD28/CTLA-4family of T cell regulators and negatively regulates T cell receptor(TCR) signals, thereby negatively regulating immune responses. PD-1 cannegatively regulated CD8+ T cell responses, and thus inhibitCD8-mediated cytotoxicity and enhance tumor growth.

PD-1 has two ligands, PD-L1 and PD-L2, which are members of the B7family. PD-L1 is unregulated on macrophages and dendritic cells (DCs) inresponse to LPS and GM-CSF treatment and on T cells and B cells upon TCRand B cell receptor signaling. PD-L1 is expressed by many tumor celllines, including myelomas, mastocytomas, and melanomas.

2. Anti-Immune Checkpoint Molecule Antibody

As described above, the immune checkpoint inhibitor can be an antibody.The antibody can bind or react with an antigen (i.e., the immunecheckpoint molecule described above.) Accordingly, the antibody may beconsidered an anti-immune checkpoint molecule antibody or an immunecheckpoint molecule antibody. The antibody can be encoded by a nucleicacid sequence contained in

The antibody can include a heavy chain polypeptide and a light chainpolypeptide. The heavy chain polypeptide can include a variable heavychain (VH) region and/or at least one constant heavy chain (CH) region.The at least one constant heavy chain region can include a constantheavy chain region 1 (CH1), a constant heavy chain region 2 (CH2), and aconstant heavy chain region 3 (CH3), and/or a hinge region.

In some embodiments, the heavy chain polypeptide can include a VH regionand a CH1 region. In other embodiments, the heavy chain polypeptide caninclude a VH region, a CH1 region, a hinge region, a CH2 region, and aCH3 region.

The heavy chain polypeptide can include a complementarity determiningregion (“CDR”) set. The CDR set can contain three hypervariable regionsof the VH region. Proceeding from N-terminus of the heavy chainpolypeptide, these CDRs are denoted “CDR1,” “CDR2,” and “CDR3,”respectively. CDR1, CDR2, and CDR3 of the heavy chain polypeptide cancontribute to binding or recognition of the antigen.

The light chain polypeptide can include a variable light chain (VL)region and/or a constant light chain (CL) region. The light chainpolypeptide can include a complementarity determining region (“CDR”)set. The CDR set can contain three hypervariable regions of the VLregion. Proceeding from N-terminus of the light chain polypeptide, theseCDRs are denoted “CDR1,” “CDR2,” and “CDR3,” respectively. CDR1, CDR2,and CDR3 of the light chain polypeptide can contribute to binding orrecognition of the antigen.

The antibody may comprise a heavy chain and a light chaincomplementarity determining region (“CDR”) set, respectively interposedbetween a heavy chain and a light chain framework (“FR”) set whichprovide support to the CDRs and define the spatial relationship of theCDRs relative to each other. The CDR set may contain three hypervariableregions of a heavy or light chain V region. Proceeding from theN-terminus of a heavy or light chain, these regions are denoted as“CDR1,” “CDR2,” and “CDR3,” respectively. An antigen-binding site,therefore, may include six CDRs, comprising the CDR set from each of aheavy and a light chain V region.

The antibody can be an immunoglobulin (Ig). The Ig can be, for example,IgA, IgM, IgD, IgE, and IgG. The immunoglobulin can include the heavychain polypeptide and the light chain polypeptide. The heavy chainpolypeptide of the immunoglobulin can include a VH region, a CH1 region,a hinge region, a CH2 region, and a CH3 region. The light chainpolypeptide of the immunoglobulin can include a VL region and CL region.

Additionally, the proteolytic enzyme papain preferentially cleaves IgGmolecules to yield several fragments, two of which (the F(ab) fragments)each comprise a covalent heterodimer that includes an intactantigen-binding site. The enzyme pepsin is able to cleave IgG moleculesto provide several fragments, including the F(ab′)2 fragment, whichcomprises both antigen-binding sites. Accordingly, the antibody can bethe Fab or F(ab′)2. The Fab can include the heavy chain polypeptide andthe light chain polypeptide. The heavy chain polypeptide of the Fab caninclude the VH region and the CH1 region. The light chain of the Fab caninclude the VL region and CL region.

The antibody can be a polyclonal or monoclonal antibody. The antibodycan be a chimeric antibody, a single chain antibody, an affinity maturedantibody, a human antibody, a humanized antibody, or a fully humanantibody. The humanized antibody can be an antibody from a non-humanspecies that binds the desired antigen having one or morecomplementarity determining regions (CDRs) from the non-human speciesand framework regions from a human immunoglobulin molecule.

a. PD-1 Antibody

The anti-immune checkpoint molecule antibody can be an anti-PD-1antibody (also referred to herein as “PD-1 antibody”), a variantthereof, a fragment thereof, or a combination thereof. The PD-1 antibodycan be Nivolumab. The anti-PD-1 antibody can inhibit PD-1 activity,thereby inducing, eliciting, or increasing an immune response against atumor or cancer and decreasing tumor growth.

b. PD-L1 Antibody

The anti-immune checkpoint molecule antibody can be an anti-PD-L1antibody (also referred to herein as “PD-L1 antibody”), a variantthereof, a fragment thereof, or a combination thereof. The anti-PD-L1antibody can inhibit PD-L1 activity, thereby inducing, eliciting, orincreasing an immune response against a tumor or cancer and decreasingtumor growth.

Vector

The vaccine can comprise one or more vectors that include a heterologousnucleic acid encoding the PRAME antigen. The one or more vectors can becapable of expressing the antigen in a quantity effective to ealicit animmune response in the mammal. The vector may comprise heterologousnucleic acid encoding the antigen. The vector can have a nucleic acidsequence containing an origin of replication. The vector can be aplasmid, bacteriophage, bacterial artificial chromosome, or yeastartificial chromosome. The vector can be either a self-replication extrachromosomal vector or a vector that integrates into a host genome.

The one or more vectors can be an expression construct, which isgenerally a plasmid that is used to introduce a specific gene into atarget cell. Once the expression vector is inside the cell, the proteinthat is encoded by the gene is produced by the cellular-transcriptionand translation machinery. The plasmid is frequently engineered tocontain regulatory sequences that act as enhancer and promoter regionsand lead to efficient transcription of the gene carried on theexpression vector. The vectors of the present invention express largeamounts of stable messenger RNA and, therefore, proteins.

The vectors may have expression enhancers such as a strong promoter, astrong termination codon, adjustment of the distance between thepromoter and the cloned gene, and the insertion of a transcriptiontermination sequence and a PTIS (portable translation initiationsequence).

The vector can be a circular plasmid or a linear nucleic acid. Thecircular plasmid and linear nucleic acid are capable of directingexpression of a particular nucleotide sequence in an appropriate subjectcell. The vector can have a promoter operably linked to theantigen-encoding nucleotide sequence, which may be operably linked totermination signals. The vector can also contain sequences required forproper translation of the nucleotide sequence. The vector may comprisesequences that are required for, or enhance the efficiency of, cloningdesired fragments including, but not limited to, PRAME antigen or othercoding sequences, regulatory sequences, and selection and/or screeningmarker coding sequences, into the vector. The vector comprising thenucleotide sequence of interest may be chimeric, meaning that at leastone of its components is heterologous with respect to at least one ofits other components. The expression of the nucleotide sequence in theexpression cassette may be under the control of a constitutive promoteror of an inducible promoter, which initiates transcription only when thehost cell is exposed to some particular external stimulus. In the caseof a multicellular organism, the promoter can also be specific to aparticular tissue or organ or stage of development.

The vector can be a plasmid. The plasmid may be useful for transfectingcells with nucleic acid encoding the PRAME antigen, and the transformedhost cells are cultured and maintained under conditions whereinexpression of the antigen takes place.

The plasmid may comprise a nucleic acid sequence that encodes one ormore of the PRAME antigens disclosed herein including coding sequencesthat encode synthetic, consensus antigen capable of eliciting an immuneresponse against an antigen, fragments of such proteins, variants ofsuch proteins, fragments of variants or fusion proteins which are madeup of combinations of consensus proteins and/or fragments of consensusprotein and/or variants of consensus protein and/or fragments ofvariants consensus proteins.

A single plasmid may contain coding sequence for a single antigen,coding sequence for two antigens, coding sequence for three antigens, orcoding sequence for four antigens.

In some embodiments, a plasmid may further comprise coding sequence thatencodes CCR20 alone or as part of one these plasmids. Similarly,plasmids may further comprise coding sequences for IL-12, IL-15, and/orIL-28.

The plasmid may further comprise an initiation codon, which may beupstream of the coding sequence, and a stop codon, which may bedownstream of the coding sequence. The initiation and termination codonmay be in frame with the coding sequence.

The plasmid may also comprise a promoter that is operably linked to thecoding sequence. The promoter operably linked to the coding sequence maybe a promoter from simian virus 40 (SV40), a mouse mammary tumor virus(MMTV) promoter, a human immunodeficiency virus (HIV) promoter such asthe bovine immunodeficiency virus (BIV) long terminal repeat (LTR)promoter, a Moloney virus promoter, an avian leukosis virus (ALV)promoter, a cytomegalovirus (CMV) promoter such as the CMV immediateearly promoter, Epstein Barr virus (EBV) promoter, or a Rous sarcomavirus (RSV) promoter. The promoter may also be a promoter from a humangene such as human actin, human myosin, human hemoglobin, human musclecreatine, or human metalothionein. The promoter may also be a tissuespecific promoter, such as a muscle or skin specific promoter, naturalor synthetic. Examples of such promoters are described in U.S.Publication No. 2004/0175727, the contents of which are incorporatedherein in its entirety.

The plasmid may also comprise a polyadenylation signal, which may bedownstream of the coding sequence. The polyadenylation signal may be aSV40 polyadenylation signal, LTR polyadenylation signal, bovine growthhormone (bGH) polyadenylation signal, human growth hormone (hGH)polyadenylation signal, or human (3-globin polyadenylation signal. TheSV40 polyadenylation signal may be a polyadenylation signal from a pCEP4plasmid (Invitrogen, San Diego, Calif.).

The plasmid may also comprise an enhancer upstream of the codingsequence. The enhancer may be human actin, human myosin, humanhemoglobin, human muscle creatine or a viral enhancer such as one fromCMV, FMDV, RSV or EBV. Polynucleotide function enhancers are describedin U.S. Pat. Nos. 5,593,972; 5,962,428; and WO94/016737, the contents ofeach are fully incorporated by reference.

The plasmid may also comprise a mammalian origin of replication in orderto maintain the plasmid extrachromosomally and produce multiple copiesof the plasmid in a cell. The plasmid may be pVAXI, pCEP4 or pREP4(Invitrogen, San Diego, Calif.). The plasmid may comprise the EpsteinBarr virus origin of replication and nuclear antigen EBNA-1 codingregion, which may produce high copy episomal replication withoutintegration. The backbone of the plasmid may be pAV0242. The plasmid maybe a replication defective adenovirus type 5 (Ad5) plasmid.

The plasmid may also comprise a regulatory sequence, which may be wellsuited for gene expression in a cell harboring the plasmid. The codingsequence may comprise a codon that may allow more efficienttranscription of the coding sequence in the host cell.

The coding sequence may also comprise an immunoglobulin (Ig) leadersequence. The leader sequence may be 5′ of the coding sequence. Theconsensus antigens encoded by this sequence may comprise an N-terminalIg leader followed by a consensus antigen protein. The N-terminal Igleader may be IgE or IgG.

The plasmid may be pSE420 (Invitrogen, San Diego, Calif.), which may beused for protein production in Escherichia coli (E. coli). The plasmidmay also be pYES2 (Invitrogen, San Diego, Calif.), which may be used forprotein production in Saccharomyces cerevisiae strains of yeast. Theplasmid may also be of the MAXBAC™ complete baculovirus expressionsystem (Invitrogen, San Diego, Calif.), which may be used for proteinproduction in insect cells. The plasmid may also be pcDNA I or pcDNA3(Invitrogen, San Diego, Calif.), which may be used for proteinproduction in mammalian cells such as Chinese hamster ovary (CHO) cells.

The vector may be circular plasmid, which may transform a target cell byintegration into the cellular genome or exist extrachromosomally (e.g.,autonomous replicating plasmid with an origin of replication).

The vector can be pVAX, pcDNA3.0, or provax, or any other expressionvector capable of expressing DNA encoding the antigen and enabling acell to translate the sequence to an antigen that is recognized by theimmune system.

Also provided herein is a linear nucleic acid vaccine, or linearexpression cassette (“LEC”), that is capable of being efficientlydelivered to a subject via electroporation and expressing one or moredesired antigens. The LEC may be any linear DNA devoid of any phosphatebackbone. The DNA may encode one or more antigens. The LEC may contain apromoter, an intron, a stop codon, and/or a polyadenylation signal. Theexpression of the antigen may be controlled by the promoter. The LEC maynot contain any antibiotic resistance genes and/or a phosphate backbone.The LEC may not contain other nucleic acid sequences unrelated to thedesired antigen gene expression.

The LEC may be derived from any plasmid capable of being linearized. Theplasmid may be capable of expressing the antigen. The plasmid can be pNP(Puerto Rico/34) or pM2 (New Caledonia/99). The plasmid may be WLV009,pVAX, pcDNA3.0, or provax, or any other expression vector capable ofexpressing DNA encoding the antigen and enabling a cell to translate thesequence to an antigen that is recognized by the immune system.

The LEC can be perM2. The LEC can be perNP. perNP and perMR can bederived from pNP (Puerto Rico/34) and pM2 (New Caledonia/99),respectively.

The vector may have a promoter. A promoter may be any promoter that iscapable of driving gene expression and regulating expression of theisolated nucleic acid. Such a promoter is a cis-acting sequence elementrequired for transcription via a DNA dependent RNA polymerase, whichtranscribes the antigen sequence described herein. Selection of thepromoter used to direct expression of a heterologous nucleic aciddepends on the particular application. The promoter may be positionedabout the same distance from the transcription start in the vector as itis from the transcription start site in its natural setting. However,variation in this distance may be accommodated without loss of promoterfunction.

The promoter may be operably linked to the nucleic acid sequenceencoding the antigen and signals required for efficient polyadenylationof the transcript, ribosome binding sites, and translation termination.

The promoter may be a CMV promoter, SV40 early promoter, SV40 laterpromoter, metallothionein promoter, murine mammary tumor virus promoter,Rous sarcoma virus promoter, polyhedrin promoter, or another promotershown effective for expression in eukaryotic cells.

The vector may include an enhancer and an intron with functional splicedonor and acceptor sites. The vector may contain a transcriptiontermination region downstream of the structural gene to provide forefficient termination. The termination region may be obtained from thesame gene as the promoter sequence or may be obtained from differentgenes.

Methods of Preparing the Vector

Provided herein are methods for preparing the vector that comprises thenucleic acid molecules encoding a PRAME antigen discussed herein. Thevector, after the final subcloning step into the mammalian expressionplasmid, can be used to inoculate a cell culture in a large scalefermentation tank, using known methods in the art.

The vector for use with the EP devices, which are described below inmore detail, can be formulated or manufactured using a combination ofknown devices and techniques, but preferably they are manufactured usingan optimized plasmid manufacturing technique that is described in alicensed, co-pending U.S. application Ser. No. 12/126,611, filed on May23, 2008. In some examples, the PRAME antigen-encoding nucleic acidmolecules used in these studies can be formulated at concentrationsgreater than or equal to 10 mg/mL. The manufacturing techniques alsoinclude or incorporate various devices and protocols that are commonlyknown to those of ordinary skill in the art, in addition to thosedescribed in U.S. Application No. 60/939,792, including those describedin a licensed patent, U.S. Pat. No. 7,238,522, issued on Jul. 3, 2007.The above-referenced application and patent, U.S. Application No.60/939,792 and U.S. Pat. No. 7,238,522, respectively, are herebyincorporated in their entirety.

Excipients and Other Components of the Vaccine

The vaccine may further comprise a pharmaceutically acceptableexcipient. The pharmaceutically acceptable excipient can be functionalmolecules such as vehicles, carriers, or diluents. The pharmaceuticallyacceptable excipient can be a transfection facilitating agent, which caninclude surface active agents, such as immune-stimulating complexes(ISCOMS), Freunds incomplete adjuvant, LPS analog includingmonophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles suchas squalene and squalene, hyaluronic acid, lipids, liposomes, calciumions, viral proteins, polyanions, polycations, or nanoparticles, orother known transfection facilitating agents.

The transfection facilitating agent is a polyanion, polycation,including poly-L-glutamate (LGS), or lipid. The transfectionfacilitating agent is poly-L-glutamate, and the poly-L-glutamate may bepresent in the vaccine at a concentration less than 6 mg/ml. Thetransfection facilitating agent may also include surface active agentssuch as immune-stimulating complexes (ISCOMS), Freunds incompleteadjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides,quinone analogs and vesicles such as squalene and squalene, andhyaluronic acid may also be used administered in conjunction with thegenetic construct. The DNA plasmid vaccines may also include atransfection facilitating agent such as lipids, liposomes, includinglecithin liposomes or other liposomes known in the art, as aDNA-liposome mixture (see for example WO9324640), calcium ions, viralproteins, polyanions, polycations, or nanoparticles, or other knowntransfection facilitating agents. The transfection facilitating agent isa polyanion, polycation, including poly-L-glutamate (LGS), or lipid.Concentration of the transfection agent in the vaccine is less than 4mg/ml, less than 2 mg/ml, less than 1 mg/ml, less than 0.750 mg/ml, lessthan 0.500 mg/ml, less than 0.250 mg/ml, less than 0.100 mg/ml, lessthan 0.050 mg/ml, or less than 0.010 mg/ml.

The pharmaceutically acceptable excipient can be one or more adjuvants.The adjuvant can be other genes that are expressed in an alternativeplasmid or are delivered as proteins in combination with the plasmidabove in the vaccine. The one or more adjuvants may be selected from thegroup consisting of: CCL20, α-interferon (IFN-α), β-interferon (IFN-β),y-interferon, platelet derived growth factor (PDGF), TNFα, TNFβ, GM-CSF,epidermal growth factor (EGF), cutaneous T cell-attracting chemokine(CTACK), epithelial thymus-expressed chemokine (TECK),mucosae-associated epithelial chemokine (MEC), MHC, CD80, CD86, IL-1,IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-15, IL-18, IL-28, IL-33, MCP-1,MIP-la, MIP-1-, IL-8, L-selectin, P-selectin, E-selectin, CD34,GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2,ICAM-3, CD2, LFA-3, M-CSF, G-CSF, mutant forms of IL-18, CD40, CD40L,vascular growth factor, fibroblast growth factor, IL-7, nerve growthfactor, vascular endothelial growth factor, Fas, TNF receptor, Flt,Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DRS, KILLER,TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38,p65Rel, MyD88, IRAK, TRAF6, IkB, Inactive NIK, SAP K, SAP-I, JNK,interferon response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5,TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA,MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAPI, TAP2, IL-15 having thesignal sequence or coding sequence that encodes the signal sequencedeleted and optionally including a different signal peptide such as thatfrom IgE or coding sequence that encodes a different signal peptide suchas that from IgE, and functional fragments thereof, or a combinationthereof. The adjuvant can be IL-12, IL-15, IL-28, IL-33, CTACK, TECK,platelet derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermalgrowth factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18,or a combination thereof.

In some embodiments adjuvant may be one or more proteins and/or nucleicacid molecules that encode proteins selected from the group consistingof: CCL-20, IL-12, IL-15, IL-28, IL-33, CTACK, TECK, MEC or RANTES.Examples of IL-12 constructs and sequences are disclosed in PCTApplication No. PCT/US1997/019502 and corresponding U.S. applicationSer. No. 08/956,865, and PCT Application No. PCT/US2012/069017, filedDec. 11, 2012, and corresponding U.S. application Ser. No. 14/365,086,filed Jun. 12, 2014, filed Dec. 12, 2011, and Ser. No. 15/055,002, filedFeb. 26, 2016, which are each incorporated herein by reference. Examplesof IL-15 constructs and sequences are disclosed in PCT Application No.PCT/US04/18962 and corresponding U.S. application Ser. No. 10/560,650,and in PCT Application No. PCT/US07/00886 and corresponding U.S.application Ser. No. 12/160,766, and in PCT Application No.PCT/US10/048827, which are each incorporated herein by reference.Examples of IL-28 constructs and sequences are disclosed in PCTApplication No. PCT/US09/039648 and corresponding U.S. application Ser.No. 12/936,192, which are each incorporated herein by reference.Examples of RANTES and other constructs and sequences are disclosed inPCT Application No. PCT/US1999/004332 and corresponding U.S. applicationSer. No. 09/622,452, which are each incorporated herein by reference.Other examples of RANTES constructs and sequences are disclosed in PCTApplication No. PCT/US11/024098, which is incorporated herein byreference. Examples of RANTES and other constructs and sequences aredisclosed in PCT Application No. PCT/US1999/004332 and correspondingU.S. application Ser. No. 09/622,452, which are each incorporated hereinby reference. Other examples of RANTES constructs and sequences aredisclosed in PCT Application No. PCT/US11/024098, which is incorporatedherein by reference. Examples of chemokines CTACK, TECK and MECconstructs and sequences are disclosed in PCT Application No.PCT/US2005/042231 and corresponding U.S. application Ser. No.11/719,646, which are each incorporated herein by reference. Examples ofOX40 and other immunomodulators are disclosed in U.S. application Ser.No. 10/560,653, which is incorporated herein by reference. ExamplesofDR5 and other immunomodulators are disclosed in U.S. application Ser.No. 09/622,452, which is incorporated herein by reference.

Other genes that can be useful as adjuvants include those encoding:MCP-1, MIP-la, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin,CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, p150.95, PECAM, ICAM-1,ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18,CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7,IL-22, nerve growth factor, vascular endothelial growth factor, Fas, TNFreceptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF,DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-1,Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, Inactive NIK, SAP K,SAP-1, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec,TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND,NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2 andfunctional fragments thereof.

The vaccine may further comprise a genetic vaccine facilitator agent asdescribed in U.S. Application No. 021,579, filed Apr. 1, 1994, which isfully incorporated by reference.

The vaccine may comprise the antigen and plasmids at quantities of fromabout 1 nanogram to 100 milligrams; about 1 microgram to about 10milligrams; or preferably about 0.1 microgram to about 10 milligrams; ormore preferably about 1 milligram to about 2 milligram. In somepreferred embodiments, vaccine according to the present inventioncomprise about 5 nanogram to about 1000 micrograms of a nucleic acidmolecule. In some preferred embodiments, vaccine can contain about 10nanograms to about 800 micrograms of a nucleic acid molecule. In somepreferred embodiments, the vaccine can contain about 0.1 to about 500micrograms of a nucleic acid molecule. In some preferred embodiments,the vaccine can contain about 1 to about 350 micrograms of a nucleicacid molecule. In some preferred embodiments, the vaccine can containabout 25 to about 250 micrograms, from about 100 to about 200 microgram,from about 1 nanogram to 100 milligrams; from about 1 microgram to about10 milligrams; from about 0.1 microgram to about 10 milligrams; fromabout 1 milligram to about 2 milligram, from about 5 nanogram to about1000 micrograms, from about 10 nanograms to about 800 micrograms, fromabout 0.1 to about 500 micrograms, from about 1 to about 350 micrograms,from about 25 to about 250 micrograms, from about 100 to about 200microgram of the antigen or plasmid thereof.

The vaccine can be formulated according to the mode of administration tobe used. An injectable vaccine pharmaceutical composition can besterile, pyrogen free and particulate free. An isotonic formulation orsolution can be used. Additives for isotonicity can include sodiumchloride, dextrose, mannitol, sorbitol, and lactose. The vaccine cancomprise a vasoconstriction agent. The isotonic solutions can includephosphate buffered saline. Vaccine can further comprise stabilizersincluding gelatin and albumin. The stabilizers can allow the formulationto be stable at room or ambient temperature for extended periods oftime, including LGS or polycations or polyanions.

Pharmaceutical Compositions of the Vaccine

The vaccine can be in the form of a pharmaceutical composition. Thepharmaceutical composition can comprise the vaccine. The pharmaceuticalcompositions can comprise about 5 nanograms (ng) to about 10 milligrams(mg) of the nucleic acid molecule of the vaccine. In some embodiments,pharmaceutical compositions according to the present invention compriseabout 25 ng to about 5 mg of the nucleic acid molecule of the vaccine.In some embodiments, the pharmaceutical compositions contain about 50 ngto about 1 mg of the nucleic acid molecule of the vaccine. In someembodiments, the pharmaceutical compositions contain about 0.1 to about500 micrograms of the nucleic acid molecule of the vaccine. In someembodiments, the pharmaceutical compositions contain about 1 to about350 micrograms of the nucleic acid molecule of the vaccine. In someembodiments, the pharmaceutical compositions contain about 5 to about250 micrograms of the nucleic acid molecule of the vaccine. In someembodiments, the pharmaceutical compositions contain about 10 to about200 micrograms of the nucleic acid molecule of the vaccine. In someembodiments, the pharmaceutical compositions contain about 15 to about150 micrograms of the nucleic acid molecule of the vaccine. In someembodiments, the pharmaceutical compositions contain about 20 to about100 micrograms of the nucleic acid molecule of the vaccine. In someembodiments, the pharmaceutical compositions contain about 25 to about75 micrograms of the nucleic acid molecule of the vaccine. In someembodiments, the pharmaceutical compositions contain about 30 to about50 micrograms of the nucleic acid molecule of the vaccine. In someembodiments, the pharmaceutical compositions contain about 35 to about40 micrograms of the nucleic acid molecule of the vaccine. In someembodiments, the pharmaceutical compositions contain about 100 to about200 micrograms of the nucleic acid molecule of the vaccine. In someembodiments, the pharmaceutical compositions comprise about 10micrograms to about 100 micrograms of the nucleic acid molecule of thevaccine. In some embodiments, the pharmaceutical compositions compriseabout 20 micrograms to about 80 micrograms of the nucleic acid moleculeof the vaccine. In some embodiments, the pharmaceutical compositionscomprise about 25 micrograms to about 60 micrograms of the nucleic acidmolecule of the vaccine. In some embodiments, the pharmaceuticalcompositions comprise about 30 ng to about 50 micrograms of the nucleicacid molecule of the vaccine. In some embodiments, the pharmaceuticalcompositions comprise about 35 ng to about 45 micrograms of the nucleicacid molecule of the vaccine. In some preferred embodiments, thepharmaceutical compositions contain about 0.1 to about 500 micrograms ofthe nucleic acid molecule of the vaccine. In some preferred embodiments,the pharmaceutical compositions contain about 1 to about 350 microgramsof the nucleic acid molecule of the vaccine. In some preferredembodiments, the pharmaceutical compositions contain about 25 to about250 micrograms of the nucleic acid molecule of the vaccine. In somepreferred embodiments, the pharmaceutical compositions contain about 100to about 200 micrograms of the nucleic acid molecule of the vaccine.

In some embodiments, pharmaceutical compositions according to thepresent invention comprise at least 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 ng of the nucleic acidmolecule of the vaccine. In some embodiments, the pharmaceuticalcompositions can comprise at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125,130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195,200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265,270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335,340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405,410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475,480, 485, 490, 495, 500, 605, 610, 615, 620, 625, 630, 635, 640, 645,650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715,720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785,790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855,860, 865, 870, 875, 880, 885, 890, 895. 900, 905, 910, 915, 920, 925,930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995 or1000 micrograms of DNA of the vaccine. In some embodiments, thepharmaceutical composition can comprise at least 1.5, 2, 2.5, 3, 3.5, 4,4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 mg or more of thenucleic acid molecule of the vaccine.

In other embodiments, the pharmaceutical composition can comprise up toand including 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95 or 100 ng of the nucleic acid molecule of the vaccine. Insome embodiments, the pharmaceutical composition can comprise up to andincluding 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150,155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220,225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290,295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360,365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430,435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500,605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670,675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740,745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810,815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880,885, 890, 895. 900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950,955, 960, 965, 970, 975, 980, 985, 990, 995, or 1000 micrograms of thenucleic acid molecule of the vaccine. In some embodiments, thepharmaceutical composition can comprise up to and including 1.5, 2, 2.5,3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 mg of thenucleic acid molecule of the vaccine.

The pharmaceutical composition can further comprise other agents forformulation purposes according to the mode of administration to be used.In cases where pharmaceutical compositions are injectable pharmaceuticalcompositions, they are sterile, pyrogen free and particulate free. Anisotonic formulation is preferably used. Generally, additives forisotonicity can include sodium chloride, dextrose, mannitol, sorbitoland lactose. In some cases, isotonic solutions such as phosphatebuffered saline are preferred. Stabilizers include gelatin and albumin.In some embodiments, a vasoconstriction agent is added to theformulation.

The vaccine can further comprise a pharmaceutically acceptableexcipient. The pharmaceutically acceptable excipient can be functionalmolecules such as vehicles, adjuvants, carriers, or diluents. Thepharmaceutically acceptable excipient can be a transfection facilitatingagent.

In some embodiments, the transfection facilitating agent is a polyanion,polycation, including poly-L-glutamate (LGS), or lipid. In oneembodiment, the transfection facilitating agent is poly-L-glutamate, andmore preferably, the poly-L-glutamate is present in the vaccine at aconcentration less than 6 mg/ml. The transfection facilitating agent canalso include surface active agents such as immune-stimulating complexes(ISCOMS), Freunds incomplete adjuvant, LPS analog includingmonophosphoryl lipid A, muramyl peptides, quinone analogs and vesiclessuch as squalene and squalene, and hyaluronic acid can also be usedadministered in conjunction with the genetic construct. In someembodiments, the transfection facilitating agent can comprise lipids,liposomes, including lecithin liposomes or other liposomes known in theart, as a DNA-liposome mixture (see for example WO/9324640), calciumions, viral proteins, polyanions, polycations, or nanoparticles, orother known transfection facilitating agents. Concentration of thetransfection agent in the vaccine can be less than 4 mg/ml, less than 2mg/ml, less than 1 mg/ml, less than 0.750 mg/ml, less than 0.500 mg/ml,less than 0.250 mg/ml, less than 0.100 mg/ml, less than 0.050 mg/ml, orless than 0.010 mg/ml.

Methods of Vaccination

Provided herein are methods for treating and/or preventingPRAME-expressing cancer using the pharmaceutical formulations describedabove. Also described herein are methods of using the pharmaceuticalformulations described above in the treatment and/or prevention ofPRAME-expressing cancer in a subject. Also described herein are methodsof vaccinating a subject. Also described herein are methods ofadministering the pharmaceutical formulations described herein to asubject in need thereof. The methods described herein collectivelyreferred to as methods of treatment using the pharmaceuticalformulations described herein can comprise administering one or morevaccine as described herein to a subject in need thereof to induce atherapeutic and/or prophylactic immune response. The vaccine can beadministered to a subject to modulate the activity of the subject'simmune system and enhance the immune response. The administration of thevaccine can be the transfection of the cancer antigens as disclosedherein as a nucleic acid molecule that is expressed in the transfectedcell and delivered to the surface of the cell, whereupon the immunesystem recognizes the antigen and induces a cellular, humoral, orcellular and humoral response. The administration of the vaccine can beused to induce or elicit an immune response in subjects against one ormore of the cancer antigens as disclosed herein by administering to thesubject the vaccine as discussed herein.

The vaccine can be administered to a subject to modulate the activity ofthe subject's immune system, thereby enhancing the immune response. Insome embodiments, the subject is a mammal. Upon administration of thevaccine to the mammal, and thereby introducing the vector into the cellsof the mammal, the transfected cells will express and secrete one ormore of the cancer antigens as disclosed herein. These secretedproteins, or synthetic antigens, will be recognized as foreign by theimmune system, which will mount an immune response that can include:antibodies made against the one or more cancer antigens, and T-cellresponse specifically against the one or more cancer antigens. In someexamples, a mammal vaccinated with the vaccines discussed herein willhave a primed immune system and when challenged with the one or morecancer antigens as disclosed herein, the primed immune system will allowfor rapid clearing of subsequent cancer antigens as disclosed herein,whether through the humoral, cellular, or both cellular and humoralimmune responses.

Methods of administering the nucleic acid molecule of a vaccine aredescribed in U.S. Pat. Nos. 4,945,050 and 5,036,006, both of which areincorporated herein in their entirety by reference.

The vaccine can be administered to a mammal to elicit an immune responsein a mammal. The mammal can be human, non-human primate, cow, pig,sheep, goat, antelope, bison, water buffalo, bovids, deer, hedgehogs,elephants, llama, alpaca, mice, rats, and preferably human, cow, or pig.The vaccine can likewise be administered to a non-mammal subject, forexample, a chicken, to elicit an immune response.

The vaccine dose can be between 1 microgram and 10 mg active componentper kilogram (kg) body weight over time (component/kg body weight/time),and can be 20 micrograms to 10 mg component/kg body weight/time. Thevaccine can be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,or 31 days. The number of vaccine doses for effective treatment can be1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more doses.

Methods of Generating an Immune Response with the Vaccine

The vaccine can be used to generate an immune response in a mammal ornon-mammal subject, including therapeutic or prophylactic immuneresponse. The immune response can generate antibodies and/or killer Tcells which are directed to the one or more cancer antigens as disclosedherein. Such antibodies and T cells can be isolated.

Some embodiments provide methods of generating immune responses againstone or more of the cancer antigens as disclosed herein, whichembodiments comprise administering the vaccine to a subject. Someembodiments provide methods of prophylactically vaccinating a subjectagainst a cancer or tumor expressing one or more of the cancer antigensas described above, which embodiments comprise administering thevaccine. Some embodiments provide methods of therapeutically vaccinatinga subject that has been suffering from the cancer or tumor expressingone or more of the cancer antigens, which embodiments compriseadministering the vaccine. Diagnosis of the cancer or tumor expressingthe one or more cancer antigens as disclosed herein prior toadministration of the vaccine can be done routinely.

Methods of Cancer Treatment with the Vaccine

The vaccine can be used to generate or elicit an immune response in amammal or subject in need thereof that is reactive or directed to anHPV-mediated PRAME-expressing cancer such as but not limited to ovariancancer, and specifically, epithelial ovarian cancer. The elicited immuneresponse can prevent cancer or tumor growth. The elicited immuneresponse can prevent and/or reduce metastasis of cancerous or tumorcells. Accordingly, the vaccine can be used in a method that treatsand/or prevents cancer or tumors in the mammal or subject administeredthe vaccine.

In some embodiments, the administered vaccine can mediate clearance orprevent growth of tumor cells by inducing (1) humoral immunity via Bcell responses to generate antibodies that block monocytechemoattractant protein-1 (MCP-1) production, thereby retarding myeloidderived suppressor cells (MDSCs) and suppressing tumor growth; (2)increase cytotoxic T lymphocyte such as CD8⁺ (CTL) to attack and killtumor cells; (3) increase T helper cell responses; (4) and increaseinflammatory responses via IFN-γ and TFN-α or preferably all of theaforementioned.

In some embodiments, the immune response can generate a humoral immuneresponse and/or an antigen-specific cytotoxic T lymphocyte (CTL)response that does not cause damage to or inflammation of varioustissues or systems (e.g., brain or neurological system, etc.) in thesubject administered the vaccine.

In some embodiments, the administered vaccine can increase tumor freesurvival, reduce tumor mass, or a combination thereof in the subject.The administered vaccine can increase tumor free survival by 20%, 21%,22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%,36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%,50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, and 60% in thesubject. The administered vaccine can reduce tumor mass by 20%, 21%,22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%,36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%,50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, and 70% in the subject after immunization.The administered vaccine can prevent and block PRAME-mediated inhibitionof retinoic acid receptor.

In some embodiments, the vaccine can be administered to the periphery(as described in more detail below) to establish an antigen-specificimmune response targeting the cancerous or tumor cells or tissue toclear or eliminate the cancer or tumor expressing the one or more cancerantigens without damaging or causing illness or death in the subjectadministered the vaccine.

The administered vaccine can increase a cellular immune response in thesubject by about 50-fold to about 6000-fold, about 50-fold to about5500-fold, about 50-fold to about 5000-fold, about 50-fold to about4500-fold, about 100-fold to about 6000-fold, about 150-fold to about6000-fold, about 200-fold to about 6000-fold, about 250-fold to about6000-fold, or about 300-fold to about 6000-fold. In some embodiments,the administered vaccine can increase the cellular immune response inthe subject by about 50-fold, 100-fold, 150-fold, 200-fold, 250-fold,300-fold, 350-fold, 400-fold, 450-fold, 500-fold, 550-fold, 600-fold,650-fold, 700-fold, 750-fold, 800-fold, 850-fold, 900-fold, 950-fold,1000-fold, 1100-fold, 1200-fold, 1300-fold, 1400-fold, 1500-fold,1600-fold, 1700-fold, 1800-fold, 1900-fold, 2000-fold, 2100-fold,2200-fold, 2300-fold, 2400-fold, 2500-fold, 2600-fold, 2700-fold,2800-fold, 2900-fold, 3000-fold, 3100-fold, 3200-fold, 3300-fold,3400-fold, 3500-fold, 3600-fold, 3700-fold, 3800-fold, 3900-fold,4000-fold, 4100-fold, 4200-fold, 4300-fold, 4400-fold, 4500-fold,4600-fold, 4700-fold, 4800-fold, 4900-fold, 5000-fold, 5100-fold,5200-fold, 5300-fold, 5400-fold, 5500-fold, 5600-fold, 5700-fold,5800-fold, 5900-fold, or 6000-fold.

The administered vaccine can increase interferon gamma (IFN-γ) levels inthe subject by about 50-fold to about 6000-fold, about 50-fold to about5500-fold, about 50-fold to about 5000-fold, about 50-fold to about4500-fold, about 100-fold to about 6000-fold, about 150-fold to about6000-fold, about 200-fold to about 6000-fold, about 250-fold to about6000-fold, or about 300-fold to about 6000-fold. In some embodiments,the administered vaccine can increase IFN-γ levels in the subject byabout 50-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold,350-fold, 400-fold, 450-fold, 500-fold, 550-fold, 600-fold, 650-fold,700-fold, 750-fold, 800-fold, 850-fold, 900-fold, 950-fold, 1000-fold,1100-fold, 1200-fold, 1300-fold, 1400-fold, 1500-fold, 1600-fold,1700-fold, 1800-fold, 1900-fold, 2000-fold, 2100-fold, 2200-fold,2300-fold, 2400-fold, 2500-fold, 2600-fold, 2700-fold, 2800-fold,2900-fold, 3000-fold, 3100-fold, 3200-fold, 3300-fold, 3400-fold,3500-fold, 3600-fold, 3700-fold, 3800-fold, 3900-fold, 4000-fold,4100-fold, 4200-fold, 4300-fold, 4400-fold, 4500-fold, 4600-fold,4700-fold, 4800-fold, 4900-fold, 5000-fold, 5100-fold, 5200-fold,5300-fold, 5400-fold, 5500-fold, 5600-fold, 5700-fold, 5800-fold,5900-fold, or 6000-fold.

The vaccine dose can be between 1 microgram and 10 mg active componentper kilogram (kg) body weight over time (component/kg body weight/time),and can be 20 micrograms to 10 mg component/kg body weight/time. Thevaccine can be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,or 31 days. The number of vaccine doses for effective treatment can be1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more doses.

Routes of Administration

The vaccine or pharmaceutical composition can be administered bydifferent routes including orally, parenterally, sublingually,transdermally, rectally, transmucosally, topically, via inhalation, viabuccal administration, intrapleurally, intravenously, intraarterially,intraperitoneally, subcutaneously, intramuscularly, intranasalintrathecally, and/or intraarticularly, or combinations thereof. Forveterinary use, the composition can be administered as a suitablyacceptable formulation in accordance with normal veterinary practice.The veterinarian can readily determine the dosing regimen and route ofadministration that is most appropriate for a particular animal. Thevaccine can be administered by traditional syringes, needlelessinjection devices, “microprojectile bombardment gene guns”, or otherphysical methods such as electroporation (“EP”), “hydrodynamic method”,or ultrasound.

The vector of the vaccine can be administered to the mammal by severalwell-known technologies including DNA injection (also referred to as DNAvaccination) with and without in vivo electroporation, liposome mediatedtransfection, nanoparticle facilitated transfection, and use recombinantvectors such as recombinant adenovirus, recombinant adenovirusassociated virus, and recombinant vaccinia. The one or more cancerantigens of the vaccine can be administered via DNA injection along within vivo electroporation.

Electroporation

The vaccine or pharmaceutical composition can be administered byelectroporation. Administration of the vaccine via electroporation canbe accomplished using electroporation devices that can be configured todeliver to a desired tissue of a mammal a pulse of energy effective tocause reversible pores to form in cell membranes, and preferably thepulse of energy is a constant current similar to a preset current inputby a user. The electroporation device can comprise an electroporationcomponent and an electrode assembly or handle assembly. Theelectroporation component can include and incorporate one or more of thevarious elements of the electroporation devices, including: controller,current waveform generator, impedance tester, waveform logger, inputelement, status reporting element, communication port, memory component,power source, and power switch. The electroporation can be accomplishedusing an in vivo electroporation device, for example CELLECTRA® EPsystem (Inovio Pharmaceuticals, Inc., Blue Bell, Pa.) or Elgenelectroporator (Inovio Pharmaceuticals, Inc.) to facilitate transfectionof cells by the plasmid.

Examples of electroporation devices and electroporation methods that canfacilitate administration of the DNA vaccines of the present inventioninclude those described in U.S. Pat. No. 7,245,963; U.S. Publication No.2005/0052630, the contents of which are hereby incorporated by referencein their entirety. Other electroporation devices and electroporationmethods that can be used for facilitating administration of the DNAvaccines include those provided in co-pending and co-owned U.S. patentapplication Ser. No. 11/874,072, filed Oct. 17, 2007, U.S. ApplicationsNos. 60/852,149, filed Oct. 17, 2006, and 60/978,982, filed Oct. 10,2007, all of which are hereby incorporated in their entirety.

U.S. Pat. No. 7,245,963 describes modular electrode systems and theiruse for facilitating the introduction of a biomolecule into cells of aselected tissue in a body or plant. The modular electrode systems cancomprise a plurality of needle electrodes; a hypodermic needle; anelectrical connector that provides a conductive link from a programmableconstant-current pulse controller to the plurality of needle electrodes;and a power source. An operator can grasp the plurality of needleelectrodes that are mounted on a support structure and firmly insertthem into the selected tissue in a body or plant. The biomolecules arethen administering via the hypodermic needle into the selected tissue.The programmable constant-current pulse controller is activated andconstant-current electrical pulse is applied to the plurality of needleelectrodes. The applied constant-current electrical pulse facilitatesthe introduction of the biomolecule into the cell between the pluralityof electrodes. The entire content of U.S. Pat. No. 7,245,963 is herebyincorporated by reference in its entirety.

U. S. Publication No. 2005/0052630 describes an electroporation devicewhich can be used to effectively facilitate the introduction of abiomolecule into cells of a selected tissue in a body or plant. Theelectroporation device comprises an electro-kinetic device (“EKDdevice”) whose operation is specified by software or firmware. The EKDdevice produces a series of programmable constant-current pulse patternsbetween electrodes in an array based on user control and input of thepulse parameters, and allows the storage and acquisition of currentwaveform data. The electroporation device also comprises a replaceableelectrode disk having an array of needle electrodes, a central injectionchannel for an injection needle, and a removable guide disk. The entirecontent of U.S. Publication No. 2005/0052630 is hereby fullyincorporated by reference.

The electrode arrays and methods described in U.S. Pat. No. 7,245,963and U.S Publication No. 2005/0052630 can be adapted for deep penetrationinto not only tissues such as muscle, but also other tissues or organs.Because of the configuration of the electrode array, the injectionneedle is also inserted completely into the target organ, and theinjection is administered perpendicular to the target issue, in the areathat is pre-delineated by the electrodes. The electrodes described inU.S. Pat. No. 7,245,963 and U.S. Publication No. 2005/005263 arepreferably 20 mm long and 21 gauge.

Additionally, contemplated in some embodiments that incorporateelectroporation devices and uses thereof, there are electroporationdevices that are those described in the following patents: U.S. Pat. No.5,273,525, issued Dec. 28, 1993; U.S. Pat. No. 6,110,161, issued Aug.29, 2000; U.S. Pat. No. 6,261,281, issued Jul. 17, 2001; U.S. Pat. No.6,958,060, issued Oct. 25, 2005; and U.S. Pat. No. 6,939,862, issuedSep. 6, 2005. Furthermore, patents covering subject matter provided inU.S. Pat. No. 6,697,669, issued Feb. 24, 2004, which concernsadministration of DNA using any of a variety of devices, and U.S. Pat.No. 7,328,064 issued Feb. 5, 2008, drawn to methods of injecting DNA arecontemplated herein. The above-patents are incorporated by reference intheir entirety.

Methods of Preparing the Vaccine

Provided herein are methods for preparing the vectors included in thevaccines discussed herein. The vectors, after the final subcloning step,can be used to inoculate a cell culture in a large scale fermentationtank, using known methods in the art.

The DNA plasmids for use with the EP devices of the present inventioncan be formulated or manufactured using a combination of known devicesand techniques, but preferably they are manufactured using an optimizedplasmid manufacturing technique that is described in a U.S. PublicationNo. 2009/0004716, filed on May 23, 2007. In some examples, the DNAplasmids used in these studies can be formulated at concentrationsgreater than or equal to 10 mg/mL. The manufacturing techniques alsoinclude or incorporate various devices and protocols that are commonlyknown to those of ordinary skill in the art, in addition to thosedescribed in U.S. Application No. 60/939,792, including those describedin a licensed patent, U.S. Pat. No. 7,238,522, issued on Jul. 3, 2007.The above-referenced application and patent, U.S. Application No.60/939,792 and U.S. Pat. No. 7,238,522, respectively, are herebyincorporated in their entirety.

The present invention has multiple aspects, illustrated by the followingnon-limiting examples.

EXAMPLES

The present invention is further illustrated in the following Examples.It should be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, various modifications of the invention in addition tothose shown and described herein will be apparent to those skilled inthe art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims.

Example 1 Synthetic Consensus PRAME

In order to generate a human consensus PRAME, 10 PRAME sequences werecollected from GenBank (https://www.ncbi.nlm.nih.gov/genbank/). TheGenBank accession numbers for selected PRAME sequences are:

NP_006106.1, AFX65483.1, XP_001090516.1, AFI35054.1, AFJ71405.1,XP_003805956.1, XP_525643.2, BAK62424.1, XP_003919211.1, andXP_003919212.1.

A consensus sequence was generated using the DNASTAR® Lasergene softwarepackage (version 13.0.0.357). The sequences listed above were importedinto MegAlign and aligned using the ClustalW multiple sequence alignmentprogram. The resulting PRAME sequence shares 95.1%-95.5% homology withthe native human PRAME sequences.

Once the synthetic consensus PRAME DNA sequence was obtained, in orderto have a higher level of expression an upstream Kozak sequence and IgEleader were added to the N-terminus. Furthermore, the codon usage ofthis gene was adapted to the codon bias of Homo sapiens genes.Additionally, RNA optimization was performed: regions of very high(>80%) or very low (<30%) GC content and the cis-acting sequence motifssuch as internal TATA boxes, chi-sites and ribosomal entry sites wereavoided. To eliminate the potential function of an expressed PRAMEmolecule in retinoic acid signaling repression, two point mutations wereintroduced (L487V and L488V) in the nuclear receptor box LRELL (SEQ IDNO: 4) of PRAME, resulting in a modified synthetic consensus PRAME. Thismodified synthetic consensus PRAME protein sequence shares 94.7%-95.1%identity with human native PRAME proteins. Characteristics of thesynthetic consensus PRAME are provided in Table 1.

TABLE 1 Characteristics of Synthetic Consensus PRAME Modified SyntheticConsensus Characteristics PRAME Identity to native human PRAME94.7%-95.1% Identity to native rhesus PRAME 98.6% Identity to nativemouse PRAME 35.2% Number of amino acid mutations (vs native human) 25-27Number of inserted mutations (not consensus derived) 2 Molecular weight528 aa (58 kDa) Length of coding sequence (bp) 1584

Referring to FIG. 2, a sequence alignment of the modified consensusPRAME with five human PRAME sequences illustrates the amino aciddifferences between the sequences. The leucine to valine substitutionsat amino acid residues 487 and 488 of the modified synthetic consensusPRAME are among the highlighted amino acid differences. Percentidentities between the aligned sequences are presented in Table 2.

TABLE 2 Percent Identity of Modified Consensus PRAME with human PRAME 12 3 4 5 1 95.1 94.7 94.9 94.9 PRAMEmut-GenScript.pro 2 5.1 99.6 99.899.8 huPRAME(AAH39731.pro) 3 5.5 0.4 99.8 99.8 huPRAME(AFX65483).pro 45.3 0.2 0.2 100 huPRAME(NP_006106).pro 5 5.3 0.2 0.2 0.0huPRAME(78395).pro

Comparisons of unmodified synthetic consensus PRAME and modifiedconsensus PRAME are provided in Table 3.

TABLE 3 Comparison of Modified and Unmodified Consensus PRAME HumanSequence Mouse Sequence Macaque Sequence Consensus PRAME 95.1% to 95.5%39.8% to 42.6% 98.6% to 98.8% Modified Consensus 94.7% to 95.1% 39.8% to42.1% 98.4% to 98.6% PRAME

Referring to FIG. 3, the synthesized synthetic consensus PRAME wasdigested with BamHI and XhoI, and cloned into Inovio's expression vectorpGX0001 with the expression cassette placed under the transcriptionalcontrol of the cytomegalovirus immediate-early promoter. The resultingplasmid was designated pGX1421 and full length sequencing was performedto confirm the sequence.

pGX1421 is a DNA plasmid encoding the synthetic consensus PRAME protein.Related mRNA production is driven by a human CMV promoter (hCMVpromoter) and terminated by the bovine growth hormone 3′-endpoly-adenylation signal (bGH polyA). The pGX0001 backbone (a modifiedpVAX1 expression vector, the original pVAX1 was obtained fromThermoFisher, St. Louis, Mo.) includes the kanamycin resistance gene(KanR) and plasmid origin of replication (pUC ori) for productionpurpose. Those elements are not functional in eukaryotic cells.

Modifications were introduced into pVAX1 to create pGX0001 and areidentified based on the reported sequence of pVAX1 available fromThermoFisher Scientific. These modifications are listed below and noissues were detected regarding plasmid amplification and antigentranscription and translation.

C>G 241 in CMV promoter

C>T 1158 backbone, downstream of the bovine growth hormonepolyadenylation signal (bGH polyA)

A>−2092 backbone, downstream of the Kanamycin resistance gene

C>T 2493 in pUC origin of replication (pUC ori)

G>C 2969 in very end of pUC Ori upstream of RNASeH site

Base pairs 2, 3 and 4 are changed from ACT to CTG in backbone, upstreamof CMV promoter.

Example 2 PRAME Expression

PRAME expression was examined in cell lines transfected with a plasmidencoding the synthetic consensus PRAME (pGX1411) or a control todetermine if the synthetic antigen was expressed. Western blot analysisof the cell lines using a commercially available anti-PRAME antibody anda β-Actin control antibody shows that only those cells transfected withpGX1411 exhibited expression of PRAME protein that migrated at thecorrect molecular weight (FIG. 4). An indirect immunofluorescent assaycompared PRAME gene expression to pVAX expression in Rhabdomyosarcomacells and Hek293 cells. Referring to FIG. 5, the synthetic consensusPRAME was expressed in both cell lines while the pVAX negative controlwas not.

Example 3 Immunogenicity in Mice

Immunogenicity studies were performed in C57Bl/6 mice according to theimmunization and bleed schedule depicted in FIG. 6. Briefly, dosages of5, 10, 15, 25, and 50 μg of pGX1411 per immunization were administeredto mice at time points Week 0 (initial immunization), Week 2, Week 4,and ELISpot. Mice were bled at time points Week 0 (baseline measurement)and Week 5. A dose response study was initially performed to determinethe most effective dose, and, referring to FIG. 7, all dosesinvestigated resulted in a greater response than naïve. There was aslight increase in response with increasing dose up to 15 μg pGX1411DNA. At higher doses of 25 and 50 μg of pGX1411 DNA, the responseplateaued.

The synthetic consensus PRAME was highly immunogenic (FIG. 8A) in miceand immunodominant epitopes were identified by epitope mapping (FIG. 8Band Table 4). In brief, peptides spanning the entire consensus PRAME,each containing 15 amino acid residues overlapping by 8 amino acids wereproduced. These peptides were pooled into 3 peptide pools (Table 4).Splenocytes from naïve and pGX1411 immunized mice were stimulated withthe 3 peptide pools in mouse IFNγ ELISpot assays. The results are shownin FIG. 8A. Immunodominant epitopes were identified using a peptidematrix mapping approach (FIG. 8B). Immunodominant epitopes areunderlined in Table 4.

TABLE 4 Immunodominant epitopes identified in mice immunizedwith pGX1411 No. of Epitope- Pool Comprising Number PeptidesSequence of Epitope-Comprising Peptides Pool 1 22 PeptidesRVHSERRRLRGSIQS (SEQ ID NO: 5) - aa 1-197RLRGSIQSRYISMSV (SEQ ID NO: 6) - SRYISMSVWTSPRRL (SEQ ID NO: 7) -VWTSPRRLVELAGQS (SEQ ID NO: 8) - LVELAGQSLLKDEAL (SEQ ID NO: 9) -SLLKDEALAIAALEL (SEQ ID NO: 10) - LLPRELFPPLFMAAF (SEQ ID NO: 11) -PPLFMAAFDGRHSQT (SEQ ID NO: 12) - FDGRHSQTLKAMVQA (SEQ ID NO: 13) -TLKAMVQAWPFTCLP (SEQ ID NO: 14) - AWPFTCLPLGVLMKG (SEQ ID NO: 15) -PLGVLMKGQQLHLET (SEQ ID NO: 16) - GQQLHLETFKAVLDG (SEQ ID NO: 17) -TFKAVLDGLDVLLAQ (SEQ ID NO: 18) - GLDVLLAQEVRPRRW (SEQ ID NO: 19) -QEVRPRRWKLEVLDL (SEQ ID NO: 20) - WKLEVLDLRKNSHQD (SEQ ID NO: 21) -LRKNSHQDFWTVWSG (SEQ ID NO: 22) - DFWTVWSGNRASLYS (SEQ ID NO: 23) -GNRASLYSFPEPEAA (SEQ ID NO: 24) - SFPEPEAAQPMRKKR (SEQ ID NO: 25) -AQPMRKKRKVDGLST (SEQ ID NO: 26) - RKVDGLSTEAEQPFT (SEQ ID NO: 27) Pool 210 Peptides TPIEVLVDLSLKEGA (SEQ ID NO: 28) - aa 190-372DLSLKEGACDELFSY (SEQ ID NO: 29) - ACDELFSYLMEKVKR (SEQ ID NO: 30) -YLMEKVKRQKNVLHL (SEQ ID NO: 31) - RQKNVLHLCCKKLKI (SEQ ID NO: 32) -LCCKKLKIFAMPMQD (SEQ ID NO: 33) - IFAMPMQDIKMILKM (SEQ ID NO: 34) -DIKMILKMVQLDSIE (SEQ ID NO: 35) - EDLEVTCTWKLPTLA (SEQ ID NO: 36) -TWKLPTLAKFSPYLG (SEQ ID NO: 37) - Pool 3 15 PeptidesASATLQDLDFDECGI (SEQ ID NO: 38) - aa 365-526LDFDECGIMDDQLLV (SEQ ID NO: 39) - IMDDQLLVLLPSLSH (SEQ ID NO: 40) -VLLPSLSHCSQLTTL (SEQ ID NO: 41) - HCSQLTTLSFCGNPI (SEQ ID NO: 42) -LSFCGNPISISVLQN (SEQ ID NO: 43) - NLLHHLIGLSNLTHV (SEQ ID NO: 44) -GLSNLTHVLYPVPLE (SEQ ID NO: 45) - ESYEDVHGTLHLGRL (SEQ ID NO: 46) -GTLHLGRLAYLHARL (SEQ ID NO: 47) - LAYLHARLRELLCEL (SEQ ID NO: 48) -LRELLCELGRPSMVW (SEQ ID NO: 49) LGRPSMVWLSANPCP (SEQ ID NO: 50) -PHCGDRTFYDPEPIL (SEQ ID NO: 51) - FYDPEPILCPCFMPN (SEQ ID NO: 52)(Previously published HLA-A*02-restricted PRAME peptides: ALYVDSLFFL(SEQ ID NO: 53), VLDGLDVLL (SEQ ID NO: 54), SLYSFPEA (SEQ ID NO: 55),SLLQHILGL (SEQ ID NO: 56) and NLTHVLYPV (SEQ ID NO: 57) (Quintarelli etal 2011)).

Antigen-specific CD4+ and CD8+ T cell responses induced by pGX1411 wereassessed by intracellular cytokine staining of peptide stimulatedsplenocytes from naïve and pGX1411 treated mice. CD4+ cells from pGX1411treated mice showed a small although significant increase in IFN-γ aftertreatment with pGX1411 (FIG. 9A), whereas CD8+ cells resulted in analmost 10-fold greater significant increase (FIG. 9B). For TNF-α andCD107a+, CD8+ cells resulted in a significantly greater increase withpGX1411 treatment (FIGS. 9D and 9F), whereas for CD4+ cells there was nodifference between naïve and pGX1411 treatment (FIGS. 9C and 9E).Overall, the synthetic consensus PRAME induces both CD4+ and CD8+T-cells, with CD8+ showing a higher cytokine response.

Endpoint titers were determined in a dose response manner for pGX1411,namely 5, 25, and 50 μg, as shown in FIG. 10. Each dose examinedincreased the endpoint titer compared to naïve, although increasing dosedid not significantly increase the endpoint titer response. To determinewhether pGX1411-induced antibodies could recognize native PRAMEexpressed in human cancers the reactivity of pGX1411 in cancer wasinvestigated by immuno-histochemistry as shown in FIG. 11. Tissuesections from human cancer biopsies were stained with sera from naïve orpGX1411 treated mice. After treatment with biotinylated secondaryantibody, tissue sections were stained with diaminobenzidine withhydrogen peroxide, and counterstained with hematoxylin. Tissue stainingwith a commercial PRAME antibody was used as a comparator. Positivestaining was detected in the melanoma cancer tissue sample with thepGX1411 vaccinated mouse sera and the commercial PRAME antibody but notwith the naïve mouse sera. Therefore, vaccination with pGX1411 has theability to induce PRAME specific antibodies.

The modified synthetic consensus PRAME encoded by pGX1421 induces animmune response similar to that generated by the unmodified syntheticconsensus PRAME encoded by pGX1411. Referring to FIG. 12, this elicitedimmune response by pGX1421 is comparable to that elicited by pGX1411when investigated by ELISpot in mice. Both pGX1411 and pGX1421, at adose of 5 μg, resulted in approximately 900 SFU/10⁶ peripheral bloodmononuclear cells (PBMCs) compared to baseline levels for naïve (0SFU/10⁶ PBMCs).

Example 4 Immunogenicity in Non-Human Primates (NHP)

Immunogenicity studies were carried out in non-human primates (NHPs)according to the immunization and bleeding schedule depicted in FIG. 13.pGX1421 was delivered intramuscularly (IM) with the 5P CELLECTRA® inalternating contralateral limbs with optimized rhesus IL-12 (pGX6006) atweek 0, 4, 8, and 12 with bleeds every 2 weeks following immunization.Referring to FIGS. 14A to 14C, cellular immunogenicity was evaluated byIFNγ ELISpot, ICS, and the measured IFNγ responses revealed an overallincrease with four immunizations with pGX1421 and pGX6006. After thethird immunization, IFNγ increased to approximately 600 SFU/10⁶ PBMCscompared to baseline levels. The fourth immunization slightly boostedthe response compared to the third immunization with approximately 700SFU/10⁶ PBMCs (FIG. 14A). The individual responses are shown in FIGS.14B and 14C, with two out of six NHPs having greater than averageresponse after the third immunization. This increased to three out ofthe six NHPs having a greater than average response after the fourthimmunization. Overall, six out of six NHPs responded as determined byIFNγ ELISpot two weeks after the third immunization (week 10) comparedto week 0.

Referring to FIGS. 15A-15D, pGX1421 and pGX6006 induce minimal CD4⁺T-cell responses compared to the CD8⁺ T-cell responses. CD4⁺ T cellresponses were not seen in any NHP, but robust CD8⁺ T-cell responseswere detected by ICS in the NHPs with the highest IFNγ ELISpot responsetwo weeks after the fourth immunization, in NHP 5840 and 5967 (FIG.15C).

Example 5 Characterizing the Adjuvant Effect of pGX6006 on PRAME-InducedImmunogenicity

Fifteen NHPs were divided into three groups as shown in Table 5 todetermine the adjuvant effect of pGX6006. The immunization and bleedingschedule was the same for all three groups and is depicted in FIG. 13.

TABLE 5 Study Groups Group N Antigen Construct Adjuvant Construct 1 5pGX1421 (2.0 mg) N/A 2 5 pGX1421 (2.0 mg) pGX6006 (0.20 mg) 3 5 pGX1411(2.0 mg) pGX6006 (0.20 mg)

Referring to FIGS. 16A-16C, IL-12 significantly increased IFNγ responsesto PRAME encoded by pGX1421 two weeks PD3 (Week 10) and PD4 (Week 14).Following the third immunization, the average IFNγ SFU induced bypGX1421 alone (108±124) was significantly lower than the averageresponse induced by pGX1421 and pGX6006 together (609±597, p<0.08). IFNγresponses boosted in Group 2 following the fourth immunization (1636±999SFU, p<0.08, FIG. 16C).

Cellular immune responses induced by pGX1421 and pGX1421 in combinationwith pGX6006 were further characterized by flow cytometry two weeks PD4(Week 14, FIGS. 17A-17C). There were minimal responses detected in theCD4⁺ T-cell compartment in either group as shown in FIG. 17A. Minimalresponses were detected in the CD8⁺ T-cell compartment when pGX1421 wasadministered without pGX6006 (FIG. 17B). The majority of thePRAME-specific CD8+ T-cell population detected in the pGX1421 incombination with pGX6006 group two weeks PD4 produced both IFNγ andTNFα. The remainder of the population was predominantly positive forIFNγ production alone. The majority of the antigen-specific CD8+ T-cellsinduced by pGX1421 in combination with pGX6006 were positive for bothCD107a and Granzyme B, indicating the potential for CTL and effectorfunction (FIG. 17C).

Cellular immune responses induced by pGX1411 in combination with pGX6006and pGX1421 in combination with pGX6006 were compared by IFNγ ELISpot(FIGS. 18A-18C) and by flow cytometry two weeks PD4 (Week 14, FIG.19A-19F). PRAME-specific IFNγ responses two weeks PD3 (666±597 SFU) andPD4 (1931±2795 SFU) induced by PGX1411 in combination with pGX6006 werecomparable to pGX1421 in combination with pGX6006 (PD3: 609±597; PD4:1636±999 SFU). There was not a significant difference between IFNγELISpot responses induced by pGX1411 in combination with pGX6006 andpGX1421 in combination with pGX6006 at any time point during the study(FIG. 18C). Further characterization by flow cytometry showed a trendtoward more robust CD4⁺ T-cell responses with pGX1411 in combinationwith pGX6006 compared to pGX1421 in combination with pGX6006 two weeksPD4 (FIG. 19A). The phenotype and magnitude of responses in the CD8⁺T-cell compartment were comparable for pGX1411 in combination withpGX6006 and pGX1421 in combination with pGX6006 (FIGS. 19B and 19C).

In summary, cellular immune responses directed at PRAME were comparablefor the pGX1411 in combination with pGX6006 and pGX1421 in combinationwith pGX6006. Inclusion of IL-12 encoded by pGX6006 significantlyincreased cellular immune responses induced by pGX1421.

The nucleotide sequence (SEQ ID NO. 1) and amino acid (SEQ ID NO. 2) forsynthetic consensus Prame are presented in Table 6 and Table 7,respectively.

It is understood that the foregoing detailed description andaccompanying examples are merely illustrative and are not to be taken aslimitations upon the scope of the invention, which is defined solely bythe appended claims and their equivalents.

Various changes and modifications to the disclosed embodiments will beapparent to those skilled in the art. Such changes and modification tothe disclosed embodiments, including without limitation those relatingto the chemical structures, substituents, derivatives, intermediates,syntheses, compositions, formulations, or methods of use of theinvention, may be made without departing from the spirit and scopethereof.

TABLE 6 Synthetic Consensus PRAME DNA Coding Sequence pGX1421 SEQ ID NO.SEQUENCE 1atggactgga catggattct gttcctggtc gctgctgcta cacgggtgca ttcagagaga cgaagactgcggggctcaat tcagagtagg tacatcagta tgtcagtctg gacctcacca cggagactgg tggaactggccgggcagagc ctgctgaagg atgaggccct ggctattgcc gctctggaac tgctgccccg agagctgttccctcccctgt tcatggcagc cttcgacgga cgccacagcc agactctgaa ggctatggtc caggcatggccctttacctg cctgcctctg ggcgtgctga tgaaggggca gcagctgcat ctggagactt tcaaagcagtgctggatggc ctggacgtgc tgctggccca ggaagtgagg cctaggcgct ggaagctgga ggtcctggatctgcgcaaaa acagccacca ggacttttgg accgtgtggt ccgggaatcg ggccagtctg tactcattcccagaacccga ggctgcacag ccaatgcgga agaaaagaaa ggtggatgga ctgtccaccg aagctgagcagccttttaca ccaatcgaag tgctggtcga tctgtccctg aaagaaggcg catgcgacga gctgttctcttatctgatgg agaaggtcaa aagacagaag aacgtgctgc acctgtgctg taagaaactg aaaatctttgctatgcccat gcaggacatc aagatgattc tgaaaatggt ccagctggat tccattgaag acctggaggtcacttgtacc tggaagctgc caacactggc caaattctct ccctacctgg gacagatgat caatctgcgacggctgctgc tgtctcacat ccatgctagc tcctctatta gtcctgagaa ggaggaagag tacattgcacagtttacttc tcagttcctg agtctgcagt gcctgcaggc cctgtatgtg gatagcctgt tctttctgagaggcaggctg gaccagctgc tgcgacacgt catgaacccc ctggaaacac tgagtgtgac taattgtagactgtcagagg gcgatgtgat gcatctgagc cagtccccta acgtgagcca gctgtccgtc ctgtctctgagtggcgtgat gctgacagac gtgagccctg aaccactgca ggccctgctg gagcgagcat ctgccactctgcaggacctg gattttgacg agtgtgggat catggacgat cagctgctgg tgctgctgcc ttcactgagccactgctccc agctgaccac actgtctttc tgtgggaacc caatctccat ttctgtgctg cagaatctgctgcaccatct gattggactg agcaacctga cccatgtgct gtaccccgtc cctctggaaa gctatgaggatgtgcacgga acactgcatc tgggcaggct ggcctatctg cacgctcgcc tgcgagaagt ggtgtgcgagctgggcagac cctcaatggt gtggctgagc gccaatccat gtccccattg cggcgaccgg acattctacgaccccgaacc tattctgtgc ccctgcttca tgcctaactg ataa

TABLE 7 Synthetic Consensus PRAME Protein Sequence pGX1421 SEQ ID NO.SEQUENCE 2 MDWTWILFLVAAATRVHSERRRLRGSIQSRYISMSVWTSPRRLVELAGQSLLKDEALAIAALELLPRELFPPLFMAAFDGRHSQTLKAMVQAWPFTCLPLGVLMKGQQLHLETFKAVLDGLDVLLAQEVRPRRWKLEVLDLRKNSHQDFWTVWSGNRASLYSFPEPEAAQPMRKKRKVDGLSTEAEQPFTPIEVLVDLSLKEGACDELFSYLMEKVKRQKNVLHLCCKKLKIFAMPMQDIKMILKMVQLDSIEDLEVTCTWKLPTLAKFSPYLGQMINLRRLLLSHIHASSSISPEKEEEYIAQFTSQFLSLQCLQALYVDSLFFLRGRLDQLLRHVMNPLETLSVTNCRLSEGDVMHLSQSPNVSQLSVLSLSGVMLTDVSPEPLQALLERASATLQDLDFDECGIMDDQLLVLLPSLSHCSQLTTLSFCGNPISISVLQNLLHHLIGLSNLTHVLYPVPLESYEDVHGTLHLGRLAYLHARLREVVCELGRPSMVWLSANPCPHCGDRTFYDPEPILCPCFMPN

What is claimed is:
 1. A nucleic acid molecule comprising: a nucleicacid sequence as set forth in SEQ ID NO: 1; a nucleic acid sequence asset forth in nucleotides 55 to 1584 of SEQ ID NO: 1; a nucleic acidsequence that encodes the amino acid sequence set forth in SEQ ID NO: 2;or a nucleic acid sequence that encodes the amino acid sequence as setforth in amino acid residues 19 to 526 of SEQ ID NO:
 2. 2. A proteincomprising one or more amino acid sequences selected from the groupconsisting of: (a) an amino acid sequence as set forth in SEQ ID NO: 2;(b) an amino acid sequence that is at least 96% identical to SEQ ID NO:2, wherein the amino acid sequence comprises a valine at amino acidposition 487 and a valine at amino acid position 488 relative to SEQ IDNO: 2; (c) an amino acid sequence as set forth in amino acid residues 19to 526 of SEQ ID NO: 2; and (d) an amino acid sequence that is at least96% identical to amino acids 19 to 526 of SEQ ID NO: 2, wherein theamino acid sequence comprises a valine at amino acid position 487 and avaline at amino acid position 488 relative to SEQ ID NO:
 2. 3. A vaccinecomprising a nucleic acid molecule encoding the protein of claim
 2. 4.The vaccine of claim 3, wherein the nucleic acid molecule comprises oneor more nucleic acid sequences selected from the group consisting of:(a) a nucleic acid sequence as set forth in SEQ ID NO: 1; (b) a nucleicacid sequence that is at least 96% identical to SEQ ID NO: 1, whereinthe nucleic acid encodes a valine at amino acid position 487 and avaline at amino acid position 488 relative to SEQ ID NO: 2; (c) anucleic acid sequence as set forth in nucleotides 55 to 1584 of SEQ IDNO: 1; and (d) a nucleic acid sequence that is at least 96% identical tonucleotides 55 to 1584 of SEQ ID NO: 1, wherein the nucleic acid encodesa valine at amino acid position 487 and a valine at amino acid position488 relative to SEQ ID NO:
 2. 5. The vaccine of claim 3 furthercomprising a pharmaceutically acceptable excipient.
 6. The vaccine ofany claim 3 further comprising an adjuvant.
 7. The vaccine of claim 6,wherein the adjuvant is IL-12, IL-15, IL-28, or RANTES.
 8. A method oftreating a subject having a cell characterized by aberrant PRAMEexpression comprising administering to the subject a therapeuticallyeffective amount of a vaccine comprising a nucleic acid moleculecomprising one or more nucleic acid sequences selected from the groupconsisting of: (a) SEQ ID NO: 1; (b) a nucleic acid sequence that is atleast 96% identical to SEQ ID NO: 1, wherein the nucleic acid encodes avaline at amino acid position 487 and a valine at amino acid position488 relative to SEQ ID NO: 2; (c) a nucleic acid sequence comprisingnucleotides 55 to 1584 of SEQ ID NO: 1; and (d) a nucleic acid sequencethat is at least 96% identical to nucleotides 55 to 1584 of SEQ ID NO:1, wherein the nucleic acid encodes a valine at amino acid position 487and a valine at amino acid position 488 relative to SEQ ID NO:
 2. 9. Amethod of treating a subject having a cell characterized by aberrantPRAME expression comprising administering to the subject atherapeutically effective amount of a vaccine comprising a nucleic acidmolecule encoding an antigen, wherein the antigen is a proteincomprising one or more amino acid sequences selected from the groupconsisting of: (a) an amino acid sequence as set forth in SEQ ID NO: 2;(b) an amino acid sequence that is at least 96% identical to SEQ ID NO:2, wherein the amino acid sequence comprises a valine at amino acidposition 487 and a valine at amino acid position 488 relative to SEQ IDNO: 2; (c) an amino acid sequence as set forth in amino acid residues 19to 526 of SEQ ID NO: 2; and (d) an amino acid sequence that is at least96% identical to amino acids 19 to 526 of SEQ ID NO: 2, wherein theamino acid sequence comprises a valine at amino acid position 487 and avaline at amino acid position 488 relative to SEQ ID NO:
 2. 10. A methodof treating cancer in a subject, the method comprising administering tothe subject a therapeutically effective amount of a vaccine comprising anucleic acid molecule comprising one or more nucleic acid sequencesselected from the group consisting of: (a) SEQ ID NO: 1; (b) a nucleicacid sequence that is at least 96% identical to SEQ ID NO: 1, whereinthe nucleic acid encodes a valine at amino acid position 487 and avaline at amino acid position 488 relative to SEQ ID NO: 2; (c) anucleic acid sequence comprising nucleotides 55 to 1584 of SEQ ID NO: 1;and (d) a nucleic acid sequence that is at least 96% identical tonucleotides 55 to 1584 of SEQ ID NO: 1, wherein the nucleic acid encodesa valine at amino acid position 487 and a valine at amino acid position488 relative to SEQ ID NO:
 2. 11. The method of claim 10 wherein thecancer is ovarian cancer.
 12. The method of claim 11, wherein the canceris epithelial ovarian cancer.
 13. The method of 11, wherein the canceris serous ovarian cancer.
 14. A method of vaccinating a subject againstcells characterized by aberrant PRAME expression comprisingadministering an amount of a vaccine effective to elicit an immuneresponse, wherein the vaccine comprises a nucleic acid moleculecomprising one or more nucleic acid sequences selected from the groupconsisting of: (a) SEQ ID NO: 1; (b) a nucleic acid sequence that is atleast 96% identical to SEQ ID NO: 1, wherein the nucleic acid encodes avaline at amino acid position 487 and a valine at amino acid position488 relative to SEQ ID NO: 2; (c) a nucleic acid sequence comprisingnucleotides 55 to 1584 of SEQ ID NO: 1; and (d) a nucleic acid sequencethat is at least 96% identical to nucleotides 55 to 1584 of SEQ ID NO:1, wherein the nucleic acid encodes a valine at amino acid position 487and a valine at amino acid position 488 relative to SEQ ID NO:
 2. 15. Amethod of vaccinating a subject against cells characterized by aberrantPRAME expression comprising administering an amount of a vaccineeffective to elicit an immune response, wherein the vaccine comprises anucleic acid molecule encoding an antigen, wherein the antigen is aprotein comprising one or more amino acid sequences selected from thegroup consisting of: (a) an amino acid sequence as set forth in SEQ IDNO: 2; (b) an amino acid sequence that is at least 96% identical to SEQID NO: 2, wherein the amino acid sequence comprises a valine at aminoacid position 487 and a valine at amino acid position 488 relative toSEQ ID NO: 2; (c) an amino acid sequence as set forth in amino acidresidues 19 to 526 of SEQ ID NO: 2; and (d) an amino acid sequence thatis at least 96% identical to amino acids 19 to 526 of SEQ ID NO: 2,wherein the amino acid sequence comprises a valine at amino acidposition 487 and a valine at amino acid position 488 relative to SEQ IDNO:
 2. 16. A method of eliciting an immune response in a subjectcomprising administering to the subject a therapeutically effectiveamount of a vaccine comprising a nucleic acid molecule comprising one ormore nucleic acid sequences selected from the group consisting of: (a)SEQ ID NO: 1; (b) a nucleic acid sequence that is at least 96% identicalto SEQ ID NO: 1, wherein the nucleic acid encodes a valine at amino acidposition 487 and a valine at amino acid position 488 relative to SEQ IDNO: 2; (c) a nucleic acid sequence comprising nucleotides 55 to 1584 ofSEQ ID NO: 1; and (d) a nucleic acid sequence that is at least 96%identical to nucleotides 55 to 1584 of SEQ ID NO: 1, wherein the nucleicacid encodes a valine at amino acid position 487 and a valine at aminoacid position 488 relative to SEQ ID NO:
 2. 17. The method of claim 16,wherein the elicited immune response is a cellular response, a humoralimmune response, or both a cellular immune response and a humoral immuneresponse.
 18. The method of claim 16, wherein the cellular immuneresponse comprises induction or secretion of interferon-gamma (IFN-γ),tumor necrosis factor-alpha (TNF-α), or both.
 19. The method of claim16, wherein the elicited immune response comprises inhibition of one ormore immune suppression factors that promote growth of a cell, tumor, orcancer expressing a PRAME antigen.
 20. The method of claim 16, whereinthe administering comprises electroporation.
 21. The method of claim 16,wherein the vaccine is administered at one or more sites on the subject.